Antibodies against epidermal growth factor receptor (EGFR) and uses thereof

ABSTRACT

Anti-EGFR antibodies, therapeutic compositions comprising combinations of anti-EGFR antibodies, as well as methods for using such antibodies and compositions to treat EGFR-related disorders (e.g., cancers), are disclosed.

RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US2012/045235, filed on Jul. 2, 2012, which claims priority toU.S. Application No. 61/504,633, filed Jul. 5, 2011. The contents of theaforementioned applications are hereby incorporated by reference.

BACKGROUND

The natural immune system has evolved to make antibodies for efficientneutralization of pathogens. Natural antibody preparations isolated fromimmunized animals are polyclonal in origin, and exhibit immunodominanceas compared to individual antibodies, which are restricted to one or afew epitopes of a particular antigen. Anti-tumor antibodies are able toblock growth or kill tumor cells to which they bind have been developedas highly effective cancer therapeutic agents. Mixtures of anti-tumorantibodies may achieve tumor suppressive effects that are greater thanachieved by any individual antibody in the mixture

Such results have been achieved by combining two or more neutralizingantibodies against the epidermal growth factor receptor, EGFR (ErbB1).Antibodies that bind to and inhibit EGFR have proven to provide usefulanti-cancer benefits and are of great medical and commercial value.Particular combinations of pairs of antagonistic, yet non-competitive,anti-EGFR antibodies resulted in downregulation of EGFR which was fasterand more effective than application of either antibody alone (Friedmanet al. (2005) PNAS 102:1915-1920). The combination of twocross-competitive (i.e., competitive with each other for binding toantigen) anti-EGFR antibodies has shown to be non-synergistic. It ispossible that binding of a plurality of antibodies to distinct epitopesof EGFR forms lattices of complexed receptors on cell surfaces, leadingto more efficient internalization and degradation than obtained withantibodies targeting a single epitope. The combination of a particularpair of anti-EGFR receptor antibodies have also been reported to resultin additive and in some cases synergistic, antitumor activity in vivo(Perera et al. (2005) Clin Cancer Res 11:6390-6399). Monoclonal antibody806, raised against the mutant de2-7 EGFR, combined with antagonisticantibody 528 displayed significantly higher anti-tumor activity in aglioma xenograft model than treatment with either antibody alone. Themechanism of the synergistic anti-tumor activity was shown to beassociated with rapid downregulation of EGFR, which was not induced bytreatment with the individual antibodies. Similarly EGFR phosphorylationwas greatly reduced in the presence of another pair of anti-EGFRantibodies, cetuximab and EMD55900 (Kamat et al. (2008) Cancer Biol Ther7:726-33).

Certain combinations of antibodies targeting the related receptor,ErbB2, have also been shown to function in synergy (Friedman et al.(2005). Trastuzumab combined with pertuzumab inhibited the survival ofBT474 breast cancer cells at doses in which individual antibodies areineffective (Nahta et al. (2004) Cancer Res 64:2343-2346). In anotherstudy three non-competitive anti-ErbB2 antibodies demonstrated far moreeffective in vitro killing of BT474 cells in combination thanindividually and similar results were obtained in a BT474 in vivoxenograft model (Spiridon et al. (2002) Clin Cancer Res 8:1699-701).

Other evidence that combining more than one antibody may enhance thegrowth suppressive (e.g., cytotoxic) effect of antibodies on tumor cellshas been reported. For example, monoclonal antibodies to the tumorantigen 17-1A were combined, tumor cell lysis was studied, and it wasfound that monoclonal antibodies, as well as combinations of competingantibodies, were ineffective, whereas combinations of two or morenon-competing antibodies resulted in complete tumor cell lysis.

In addition to combining antibodies, higher antibody potency has alsobeen achieved by increasing the antigen affinity of recombinantlyexpressed anti-tumor antibodies through recombinant DNA techniques knownas affinity maturation.

Accordingly, additional approaches and methods for producing anti-tumorantibody action so as to enhance the responsiveness of tumors toanti-EGFR antibodies and antibody combinations are still needed,including anti-EGFR antibodies with higher tumor affinity andcombinations of such high-affinity anti-EGFR antibodies that enhancesignaling inhibition and provide more effective cytostatic or cytotoxicanti-tumor outcomes.

SUMMARY

Novel monoclonal antibodies that bind to EGFR and inhibit various EGFRfunctions are provided herein. These antibodies provide usefultherapeutic effects, and when combined with each other or with otheranti-ErbB receptor antibodies (e.g., other anti-EGFR antibodies), arecapable of exhibiting a synergistic or additive therapeutic effectcompared to the administration of each antibody alone. These antibodies,when administered individually or in combinations as herein provided,are useful for treating a variety of disorders (e.g., cancers)associated with EGFR-mediated cellular signaling. Accordingly, isolatednovel monoclonal antibodies that exhibit the properties of binding toEGFR and inhibiting various EGFR functions, and combinations of suchantibodies that exhibit such properties are also provided herein. Usesof these antibodies for diagnostic and therapeutic purposes are alsoprovided, as are uses of the antibodies and antibody combinations hereindisclosed.

In one embodiment, a monoclonal antibody is provided which binds EGFRextracellular domain and comprises heavy and light chain CDR1, CDR2, andCDR3, sequences, wherein the heavy and light chain CDR1, CDR2, and CDR3,sequences are selected from the group consisting of:

(a) heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and3 respectively, and light chain CDR1, CDR2, and CDR3 sequences of SEQ IDNOs: 4, 5, and 6, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 7, 8, and9, respectively, and light chain CDR1, CDR2, and CDR3 sequences of SEQID NOs: 10, 11 and 12, respectively; and

(c) heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 13, 14,and 15, respectively, and light chain CDR1, CDR2, and CDR3 sequences ofSEQ ID NOs: 16, 17, and 18, respectively.

In another embodiment, a monoclonal antibody is provided that binds toEGFR extracellular domain and comprises a heavy chain variable regionand a light chain variable region, wherein the heavy and light chainvariable region sequences are selected from the group consisting of:

(a) a heavy chain variable region comprising SEQ ID NO: 19 and a lightchain variable region comprising SEQ ID NO: 20;

(b) a heavy chain variable region comprising SEQ ID NO: 21 and a lightchain variable region comprising SEQ ID NO: 22; and

(c) a heavy chain variable region comprising SEQ ID NO: 23 and a lightchain variable region comprising SEQ ID NO: 24.

The aforementioned monoclonal antibodies can bind to EGFR with a K_(D)of, for example, better than 100 nM, or better than 10 nM, or betterthan 1 nM, or better than 100 pM, or better than 10 pM, or better than 1pM. The monoclonal antibodies can exhibit one or more of the functionalproperties disclosed herein. The monoclonal antibody can be, forexample, a human antibody. In other embodiments, the antibody can be abispecific antibody, immunoconjugate, Fab, Fab′2, ScFv, Affibody®,avimer, nanobody or a domain antibody. In other embodiments, themonoclonal antibody can be, e.g., an IgG1, IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgAsec, IgD, or IgE isotype antibody.

Also provided are pharmaceutical compositions comprising any one or moreof the aforementioned anti-EGFR monoclonal antibodies and apharmaceutically acceptable carrier. Kits are also provided. The kit cancomprise, for example, a pharmaceutical composition in a container.Methods of treating cancer in a subject, comprising administering to thesubject an effective amount of the pharmaceutical composition comprisingany one or more of the aforementioned anti-EGFR monoclonal antibodiesare also provided. The aforementioned anti-EGFR monoclonal antibodies orcombinations thereof for the treatment of a cancer (or for manufactureof a medicament for the treatment of a cancer) are also provided.

In another embodiment, a composition comprising two or three monoclonalantibodies which bind to EGFR extracellular domain is provided, whereinthe two or three monoclonal antibodies are selected from the groupconsisting of:

(a) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;

(b) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively and

(c) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 13, 14, and 15, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17 and 18,respectively;

and wherein the composition comprises (a) and (b), (a) and (c), (b) and(c) or (a), (b) and (c).

In yet another embodiment, a composition comprising two or threemonoclonal antibodies which bind to EGFR extracellular domain isprovided, wherein the two or three monoclonal antibodies are selectedfrom the group consisting of:

(a) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 19 and a light chain variable region comprisingSEQ ID NO: 20;

(b) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 21 and a light chain variable region comprisingSEQ ID NO: 22; and

(c) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24;

and wherein the composition comprises (a) and (b), (a) and (c), (b) and(c) or (a) (b) and (c).

Each of monoclonal antibodies (a), (b) and (c) in the aforementionedcompositions comprising two or three antibodies can bind to EGFR with aK_(D) of, for example, better than 100 nM, or better than 10 nM orbetter than 1 nM. Each of monoclonal antibodies (a), (b) and (c) canexhibit one or more of the functional properties disclosed herein. Eachof monoclonal antibodies (a), (b) and (c) can be, for example, a humanantibody. In other embodiments, one or more of monoclonal antibodies(a), (b), and (c) is independently selected from the group consisting ofa bispecific antibody, immunoconjugate, Fab, Fab′2, ScFv, Affibody®,avimer, nanobody, and a domain antibody. In other embodiments, each ofmonoclonal antibodies (a), (b), and (c) is independently selected fromthe group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec,IgD and IgE isotype antibodies. Monoclonal antibodies (a), (b) and (c)may also be in the form of IgY and camelid antibodies.

Also provided is a pharmaceutical composition comprising any one of theaforementioned compositions comprising two or three anti-EGFR monoclonalantibodies and a pharmaceutically acceptable carrier. Kits are alsoprovided. The kit can comprise, for example, a pharmaceuticalcomposition in a container. Methods of treating cancer in a subject,comprising administering to the subject an effective amount of thepharmaceutical composition comprising any one of the aforementionedcompositions comprising two or three anti-EGFR monoclonal antibodies arealso provided. The aforementioned compositions comprising two or threeanti-anti-EGFR monoclonal antibodies (and their use for the manufactureof a medicament) for the treatment of a cancer are also provided.

In another embodiment, a composition is provided comprising threemonoclonal anti-EGFR antibodies, said composition comprising a firstantibody, a second antibody and a third antibody, wherein (i) the firstantibody comprises heavy chain CDR1, CDR2, and CDR3 sequences of SEQ IDNOs: 1, 2, and 3 respectively, and light chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 4, 5, and 6, respectively; (ii) the secondantibody comprises heavy chain CDR1, CDR2, and CDR3 sequences of SEQ IDNOs: 7, 8, and 9, respectively, and light chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 10, 11 and 12, respectively; and (iii) thethird antibody comprises heavy chain CDR1, CDR2, and CDR3 sequences ofSEQ ID NOs: 13, 14, and 15 respectively, and light chain CDR1, CDR2, andCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively, and whereinthe first second and third antibodies are present at a molar ratio of2:2:1 to each other.

In yet another embodiment, a composition is provided comprising threemonoclonal anti-EGFR antibodies, said composition comprising a firstantibody, a second antibody and a third antibody, wherein (i) the firstantibody comprises a heavy chain variable region comprising SEQ ID NO:19 and a light chain variable region comprising SEQ ID NO: 20; (ii) thesecond antibody comprises a heavy chain variable region comprising SEQID NO: 21 and a light chain variable region comprising SEQ ID NO: 22;and (iii) the third antibody comprises a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24, and wherein the first second and third antibodies arepresent at a molar ratio of 2:2:1 to each other.

Each of the first, second and third antibodies in the aforementionedcompositions can bind to EGFR with a K_(D) of, for example, better than100 nM, or better than 10 nM or better than 1 nM. In another embodiment,the first antibody binds to EGFR with a K_(D) in a range of 1×10⁻⁹ M to1.1×10⁻¹¹ M, the second antibody binds to EGFR with a K_(D) in a rangeof 1×10⁻⁹ M to 7.0×10⁻¹¹ M and the third antibody binds to EGFR with aK_(D) in a range of 1×10⁻⁹ M to 3.6×10⁻¹⁰ M. Each of the first, secondand third antibodies in the aforementioned compositions can exhibit oneor more of the functional properties disclosed herein. Each of thefirst, second and third antibodies in the aforementioned compositionscan be, for example, a human antibody. In other embodiments, one or moreof the first antibody, the second antibody and the third antibody isindependently selected from the group consisting of a bispecificantibody, immunoconjugate, Fab, Fab′2, ScFv, affibody, avimer, nanobody,and a domain antibody. In other embodiments, each of the first antibody,the second antibody and the third antibody is independently selectedfrom the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,IgAsec, IgD and IgE isotype antibodies.

Also provided are pharmaceutical compositions comprising any one of theaforementioned anti-EGFR compositions comprising a first antibody, asecond antibody and a third antibody at a 2:2:1 ratio, and apharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition is a sterile composition. In anotherembodiment, the pharmaceutical composition is suitable for injection. Inyet another embodiment, the pharmaceutical composition is a sterilecomposition suitable for intravenous injection. Kits are also provided.The kit can comprise, for example, a pharmaceutical composition in acontainer. Methods of treating cancer in a subject, comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition comprising any one of the aforementioned anti-EGFRcompositions comprising first, second and third antibodies at a 2:2:1ratio are also provided. Use of any of the aforementioned anti-EGFRcompositions comprising first, second and third antibodies at a 2:2:1ratio for the manufacture of a medicament for the treatment of a cancerare also provided.

In another embodiment, a method of preparing an anti-EGFR antibodycomposition is provided, the method comprising combining in a singlecomposition:

(a) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;

(b) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively; and

(c) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 13, 14, and 15 respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17, and 18,respectively;

wherein (a), (b) and (c) are combined at a molar ratio of 2:2:1 to eachother.

In another embodiment, a method of preparing an anti-EGFR antibodycomposition is provided, the method comprising combining in a singlecomposition:

(a) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 19 and a light chain variable region comprisingSEQ ID NO:20;

(b) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 21 and a light chain variable region comprisingSEQ ID NO: 22; and

(c) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24;

wherein (a), (b) and (c) are combined at a molar ratio of 2:2:1 to eachother.

In another embodiment, a method of treating a subject with anti-EGFRantibodies is provided, the method comprising administering to thesubject:

(a) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;

(b) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively; and

(c) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 13, 14, and 15 respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17, and 18,respectively;

wherein (a), (b) and (c) are administered to the subject at a molarratio of 2:2:1 to each other.

In another embodiment, a method of treating a subject with anti-EGFRantibodies is provided, the method comprising administering to thesubject:

(a) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 19 and a light chain variable region comprisingSEQ ID NO:20;

(b) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 21 and a light chain variable region comprisingSEQ ID NO: 22; and

(c) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24;

wherein (a), (b) and (c) are administered to the subject at a molarratio of 2:2:1 to each other. In further embodiments, the method furthercomprises co-administration to the subject of an effective amount of anadditional agent selected from the group consisting of irinotecan,MM-121, and docetaxel.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a bar graph showing the results of a direct ELISA epitopebinning experiment with the P1X, P2X and P3X antibodies using wild-typeEGFR-ECD antigen (WT), a Bin 1 epitope mutant (Bin 1 MT) and a Bin 3epitope mutant (Bin 3 MT).

FIG. 1B is a graph showing the results of a surface plasmon resonanceepitope binning experiment using the ICR10 epitope (Bin 2) conjugated ona Biacore chip, with injection of wild-type EGFR-ECD antigen, followedby sequential injection of the P1X, P2X and P3X antibodies.

FIG. 2A is a graph showing the results of a surface plasmon resonanceepitope binning experiment using the P1X antibody conjugated on aBiacore chip, with injection of wild-type EGFR-ECD antigen, followed bysequential injection of the P3X, P2X and P1X antibodies.

FIG. 2B is a graph showing the results of a surface plasmon resonanceepitope binning experiment using the P2X antibody conjugated on aBiacore chip, with injection of wild-type EGFR-ECD antigen, followed bysequential injection of the P3X, P2X and P1X antibodies.

FIG. 2C is a graph showing the results of a surface plasmon resonanceepitope binning experiment using the P3X antibody conjugated on aBiacore chip, with injection of wild-type EGFR-ECD antigen, followed bysequential injection of the P3X, P2X and P1X antibodies.

FIG. 3 is a graph showing the results of P1X, P2X and P3X antibodybinding kinetics to EGFR on A431 cells as measured via flow cytometry(FACS plot).

FIG. 4 is a graph showing the results of a phospho-EGFR (pEGFR) ELISA,demonstrating pEGFR inhibition by single antibodies: single-agenttreatment with P1X, P2X or P3X antibody.

FIG. 5A is a graph showing the results of a phospho-ERK (pERK) ELISA,demonstrating the effect of affinity maturation on pERK inhibition bycomparison of pERK inhibition by parental (ca antibody) and affinitymatured P1X antibody.

FIG. 5B is a graph showing the results of a phospho-ERK (pERK) ELISA,comparing pERK inhibition by parental (ca and ch antibodies) andaffinity matured P1X+P3X antibodies.

FIG. 5C is a graph showing the results of a phospho-ERK (pERK) ELISA,comparing pERK inhibition by parental (ca and cd antibodies) andaffinity matured P1X+P2X antibodies.

FIG. 6A is a graph showing the results of a phospho-ERK (pERK) ELISA,demonstrating pERK inhibition by P1X antibody, with an IC₅₀ value ofapproximately 25 nM.

FIG. 6B is a graph showing the results of a phospho-ERK (pERK) ELISA forA431 cells treated with a dilution series of 5 combination ratios ofP3X+P2X in combination with a constant P1X concentration of 25 nM.

FIG. 6C is a graph showing the results of a phospho-ERK (pERK) ELISA forA431 cells treated with a dilution series of 6 combination ratios ofP1X:P2X:P3X.

FIG. 6D is a graph showing the results of a phospho-ERK (pERK) ELISA forA431 cells treated with a dilution series of 5 combination ratios ofP1X:P2X.

FIG. 7A is a graph showing the results of a phospho-EGFR (pEGFR) ELISA(circles) and a phospho-ERK (pERK) ELISA (triangles), demonstratinginhibition by a 2:2:1 combination of P1X:P2X:P3X.

FIG. 7B is a graph showing the results of a phospho-ERK (pERK) ELISA,demonstrating inhibition by a 2:2:1 formulation of P1X:P2X:P3X(P1X+P2X+P3X) versus P1X single antibody alone.

FIG. 7C is a graph showing the results of a phospho-ERK (pERK) ELISA,demonstrating inhibition by P1X+P2X+P3X versus P2X single antibodyalone.

FIG. 8A is a bar graph showing western blot analysis results for totalEGFR (tEGFR) internalization kinetics of H1975 cells pre-treated withP1X+P2X+P3X antibodies for various periods of time before stimulationwith EGF.

FIG. 8B is a bar graph showing western blot analysis results for H1975cells pre-treated with P1X+P2X+P3X antibodies before stimulation withEGF, showing levels of tEGFR, pERK, phospho-AKT (pAKT) and phospho-c-Jun(p-c-Jun) in the cells, normalized to the loading control (PCNA) and tolysates of control untreated cells.

FIG. 9A is a graph showing the results of a cell viability assay forHCC827 cells, demonstrating inhibition of tumor cell proliferation bytreatment with P1X+P2X+P3X antibodies, as compared to cetuximab, in thepresence of EGF ligand.

FIG. 9B is a graph showing the results of a cell viability assay forH1975 cells, demonstrating inhibition of tumor cell proliferation bytreatment with P1X+P2X+P3X antibodies, as compared to cetuximab, in thepresence of EGF ligand.

FIG. 9C is a graph showing the results of a cell viability assay forHCC827 cells, demonstrating inhibition of tumor cell proliferation bytreatment with P1X+P2X+P3X antibodies, as compared to cetuximab, in thepresence of amphiregulin (AREG) ligand.

FIG. 9D is a graph showing the results of a cell viability assay forH1975 cells, demonstrating inhibition of tumor cell proliferation bytreatment with P1X+P2X+P3X antibodies, as compared to cetuximab, in thepresence of AREG ligand.

FIG. 10A is a graph showing the results of a DU145 tumor xenograft mousemodel experiment, demonstrating decreased tumor volume in vivo in micetreated with P1X+P2X+P3X antibodies, as compared to PBS and cetuximabcontrols.

FIG. 10B is a graph showing the results of an H1975 tumor xenograftmouse model experiment, demonstrating decreased tumor volume in vivo inmice treated with P1X+P2X+P3X antibodies, as compared to PBS control.

FIG. 11 is a graph showing the results of a ligand antagonism cellbinding assay, demonstrating the EGF ligand blocking ability of P1X, P2Xor P3X alone at low doses.

FIG. 12A is a graph showing the results of a ligand antagonism cellbinding assay, demonstrating the EGF ligand blocking ability of P1X, P2Xor P3X alone, or in triple combination, at high doses.

FIG. 12B is a graph showing the results of a ligand antagonism cellbinding assay, demonstrating the EGF ligand blocking ability of P1X Fab,P2X Fab or P3X Fab alone, or in triple combination, at high doses.

FIG. 13A is a graph showing the results of a phospho-EGFR inhibitionassay, demonstrating the inhibitory ability of triple combinations ofP1X+P2X+P3X or P1X Fab+P2X Fab+P3X Fab at low doses (50 ng/ml; 8 nM).

FIG. 13B is a graph showing the results of a phospho-EGFR inhibitionassay, demonstrating the inhibitory ability of triple combinations ofP1X+P2X+P3X or P1X Fab+P2X Fab+P3X Fab at high doses (500 ng/ml; 80 nM).

FIG. 13C is a graph showing the results of a phospho-ERK inhibitionassay, demonstrating the inhibitory ability of triple combinations ofP1X+P2X+P3X or P1X Fab+P2X Fab+P3X Fab at low doses (50 ng/ml; 8 nM).

FIG. 13D is a graph showing the results of a phospho-ERK inhibitionassay, demonstrating the inhibitory ability of triple combinations ofP1X+P2X+P3X or P1X Fab+P2X Fab+P3X Fab at high doses (500 ng/ml; 80 nM).

FIG. 14 is an immunoblot of an EGF-receptor downregulation assay incells pre-treated with triple combinations of P1X+P2X+P3X or P1X Fab+P2XFab+P3X Fab or with cetuximab. The housekeeping protein pcna was used asa control.

FIG. 15 is a graph showing the results of a patient derived colorectalcancer tumor xenograft murine mouse model experiment, demonstratingdecreased tumor volume in vivo in mice treated with ca+P2X+P3Xantibodies and irinotecan alone and in combination, as compared to PBScontrol.

FIGS. 16A-N each present a set of two tables showing the results of acell viability assay for cell lines as indicated, demonstratinginhibition of tumor cell proliferation by treatment with combinations ofP1X+P2X+P3X antibodies with MM-121 antibody in the presence of EGF andHRG ligands.

FIGS. 17A-H each present a set of two tables showing the results of acell viability assay for cell lines as indicated, demonstratinginhibition of tumor cell proliferation by treatment with combinations ofP1X+P2X+P3X antibodies with docetaxel in the presence of EGF ligand.

FIGS. 18A-D each present a set of two tables showing the results of acell viability assay for cell lines as indicated, demonstratinginhibition of tumor cell proliferation by treatment with combinations ofP1X+P2X+P3X antibodies with SN-38 in the presence of EGF ligand.

DETAILED DESCRIPTION I. Definitions

The terms “EGFR,” “ErbB1,” and “EGF receptor” are used interchangeablyherein to refer to human EGFR protein; see UniProtKB/Swiss-Prot entryP00533. The amino acid sequence of the extracellular domain of humanEGFR (EGFR-ECD) is shown in Example 1 and in SEQ ID NO:33.

The term “inhibition” as used herein, refers to any statisticallysignificant decrease in biological activity, including full blocking ofthe activity. For example, “inhibition” can refer to a statisticallysignificant decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or about 100% in biological activity.

Inhibition of phosphorylation, as used herein, refers to the ability ofan antibody to statistically significantly decrease the phosphorylationof a substrate protein relative to the signaling in the absence of theantibody (control). As is known in the art, intracellular signalingpathways include, for example, phosphoinositide 3′-kinase/Akt(PI3K/Akt/PTEN or “AKT”) and/or mitogen-activated protein kinase(MAPK/ERK or “ERK”) pathways. As is also known in the art, EGFR mediatedsignaling can be measured by assaying for the level phosphorylation ofthe substrate (e.g., phosphorylation or no phosphorylation of AKT and/orERK). Accordingly, in one embodiment, anti-EGFR antibody combinationsand compositions provide statistically significant inhibition of thelevel of phosphorylation of either or both of AKT and ERK by at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90%, orabout 100% relative to the level of phosphorylation of AKT and/or ERK inthe absence of such antibody (control). Such EGFR mediated signaling canbe measured using art recognized techniques which measure a protein in acellular cascade involving EGFR, e.g., ELISA, western, or multiplexmethods, such as Luminex®.

The phrase “inhibition of the growth of cells expressing EGFR,” as usedherein, refers to the ability of an antibody to statisticallysignificantly decrease the growth of a cell expressing EGFR relative tothe growth of the cell in the absence of the antibody (control) eitherin vivo or in vitro. In one embodiment, the growth of a cell expressingEGFR (e.g., a cancer cell) may be decreased by at least 10%, or at least20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%,or at least 70%, or at least 80%, or at least 90%, or about 100% whenthe cells are contacted with an antibody composition of combinationdisclosed herein, relative to the growth measured in the absence of theantibody composition of combination (control). Cellular growth can beassayed using art recognized techniques which measure the rate of celldivision, the fraction of cells within a cell population undergoing celldivision, and/or the rate of cell loss from a cell population due toterminal differentiation or cell death (e.g., using a cell titer glowassay or thymidine incorporation).

The phrase “inhibition of an EGFR ligand binding to EGFR,” as usedherein, refers to the ability of an antibody to statisticallysignificantly decrease the binding of an EGFR ligand to its receptor,EGFR, relative to the EGFR ligand binding in the absence of the antibody(control). This means that, in the presence of the antibody, the amountof the EGFR ligand that binds to EGFR relative to a control (noantibody), is statistically significantly decreased. The amount of anEGFR ligand which binds EGFR may be decreased in the presence of anantibody composition or combination disclosed herein by at least 10%, orat least 20%, or at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90%, or about100% relative to the amount in the absence of the antibody (control). Adecrease in EGFR ligand binding can be measured using art-recognizedtechniques that measure the level of binding of labeled EGFR ligand(e.g., radiolabelled EGF or radiolabeled betacellulin) to cellsexpressing EGFR in the presence or absence (control) of the antibody.

The phrase “inhibition of EGFR dimerization,” as used herein, refers tothe ability of an antibody to statistically significantly decrease EGFRdimerization (pairing with another ErbB receptor to form homodimers,e.g., ErbB1/ErbB1 pairings, or heterodimers, e.g., ErbB1/ErbB3 pairings)relative to EGFR dimerization in the absence of the antibody (control).In one embodiment, dimerization of EGFR may be decreased by at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90%, orabout 100% when cells expressing EGFR are contacted with an antibodycomposition or combination disclosed herein, relative to dimerization ofEGFR measured in the absence of the antibody (control). A decrease inEGFR dimerization can be measured using art-recognized techniques thatmeasure the level of EGFR dimerization in the presence or absence(control) of the antibody.

The phrase “downregulation of EGFR expression,” as used herein, refersto the ability of an antibody to statistically significantly decreasethe expression of EGFR on a cell surface, for example, by increasinginternalization of EGFR and/or by decreasing recycling of EGFR fromintracellular vesicles relative to EGFR expression in the absence of theantibody (control). In one embodiment, expression of EGFR may bedecreased by at least 10%, or at least 20%, or at least 30%, or at least40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%,or at least 90%, or about 100% when cells expressing EGFR are contactedwith an antibody composition of combination provided herein, relative toexpression of EGFR on the cell surface measured in the absence of theantibody (control). Downregulation of EGFR expression on a cell surfaceincludes, for example, an increase in internalization/recycling of thereceptor, and/or an increase in internalization/degradation of thereceptor. An increase in EGFR internalization can be measured usingart-recognized techniques that measure the level of EGFR internalizationin the presence or absence (control) of the antibody.

With respect to combinations of EGFR antibodies (described herein), thewords “additive” or “additivity,” as used herein, refer to the activityof two or more antibodies wherein their combined activity (relative to aparticular function, e.g., inhibition of cell growth) is equal to thesum of their individual activities. That is, the sum of the activitiesof two or more antibodies provided herein, when acting individually on acell expressing EGFR, is approximately equivalent to the combined effectof the same antibodies acting together on the same cell. In oneembodiment, the additive effect is measured with respect to any of theproperties discussed above (e.g., inhibition of AKT or ERKphosphorylation, inhibition of the growth of cells expressing EGFR,etc.).

The words “synergy” or “synergistic,” as used herein, refer to theactivity of two or more antibodies wherein their combined activity(relative to a particular function, e.g., inhibition of cell growth) isgreater than the expected additive effect of their individualactivities. For example, the expected additive effect can be definedaccording to Bliss independence criteria. In accordance with the Blisscriteria, the effect of two or more drugs (e.g., antibodies) is equal tothe sum of the effects of the individual drugs minus the multiplicationof the effects of the individual drugs:E12=E1+E2−E1*E2where E1 is the fractional inhibition by drug 1, E2 is the fractionalinhibition by drug 2, and E12 is the expected fractional inhibition bythe combination. % inhibition is calculated by multiplying fractionalinhibition by 100%. A Bliss independence score is calculated bysubtraction of the observed % inhibition from the calculated E12%inhibition:Bliss independence score=(E12−fraction inhibition observed)*100%

The synergistic effect can apply to any of the properties discussedherein (e.g., inhibition of EGFR-dependant AKT or ERK phosphorylation,inhibition of the growth of cells expressing EGFR, etc.). In aparticular embodiment, at least 10%, or at least 20%, or at least 30%,or at least 40%, or at least 50%, or at least 60%, or at least 70%, orat least 80%, or at least 90%, or greater increase in activity of thecombined antibodies relative to the additive effect of their individualactivities is achieved.

HSA indicates highest single agent. As used herein, HSA refers to acriterion in which the activity of a combination of two drugs (e.g.antibodies) is compared to the higher of the two individual activitiesof the two drugs when used as single agents at the same doses as used inthe combination. A combination of drugs is assigned an HSA score of“True” if the observed activity of the combination is greater than thehigher activity of either of the two single drugs and “False” ifotherwise. The HSA can be measured for any of the properties discussedherein (e.g., inhibition of EGFR-dependent AKT or ERK phosphorylation,inhibition of the growth of cells expressing EGFR, etc.).

The definitions of the terms “additive,” “additivity,” “synergy,”“synergistic,” and “HSA,” as described above, also apply to acombination comprised of a combination of EGFR antibodies and anotherdrug (e.g. a chemotherapeutic agent, an antibody with specificity for aprotein other than EGFR, etc.) with the following modification: theactivity of the combination of EGFR antibodies is treated as a singulardrug (e.g. “drug 1”).

The term “antibody” or “immunoglobulin,” as used interchangeably herein,includes whole antibodies and any antigen binding fragment(antigen-binding portion) or single chain cognates thereof. An“antibody” comprises at least one heavy (H) chain and one light (L)chain. In naturally occurring IgGs, for example, these heavy and lightchains are inter-connected by disulfide bonds and there are two pairedheavy and light chains, these two also inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR) or Joining (J) regions (JH or JL in heavy and light chainsrespectively). Each V_(H) and V_(L) is composed of three CDRs three FRsand a J domain, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, J. The variableregions of the heavy and light chains bind with an antigen. The constantregions of the antibodies may mediate the binding of the immunoglobulinto host tissues or factors, including various cells of the immune system(e.g., effector cells) or humoral factors such as the first component(Clq) of the classical complement system. Thus one or more fragments ofan antibody that retain the ability to specifically bind to an antigen(e.g., EGFR) may be used in the combinations disclosed herein. It hasbeen shown that fragments of a full-length antibody can perform theantigen-binding function of an antibody. Examples of binding fragmentsdenoted as an antigen-binding portion or fragment of an antibody include(i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and CH1 domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAbfragment (Ward et al. (1989) Nature 341, 544-546), which consists of aV_(H) domain; (vii) a dAb which consists of a VH or a VL domain; and(viii) an isolated complementarity determining region (CDR) or (ix) acombination of two or more isolated CDRs which may optionally be joinedby a synthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions are paired to form monovalent molecules (such a single chaincognate of an immunoglobulin fragment is known as a single chain Fv(scFv). Such single chain antibodies are also intended to be encompassedwithin the term “antibody”. Antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same general manner as areintact antibodies. Antigen-binding portions can be produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Antigen binding fragments (including scFvs) of suchimmunoglobulins are also encompassed by the term “monoclonal antibody”as used herein. Monoclonal antibodies are highly specific, beingdirected against a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations, which typically includedifferent antibodies, directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. Monoclonal antibodies can be prepared usingany art recognized technique and those described herein such as, forexample, a hybridoma method, a transgenic animal, recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), or using phage antibodylibraries using the techniques described in, for example, U.S. Pat. No.7,388,088 and U.S. patent application Ser. No. 09/856,907 (PCT Int. Pub.No. WO 00/31246). Monoclonal antibodies include chimeric antibodies,human antibodies and humanized antibodies and may occur naturally or beproduced recombinantly.

The term “recombinant antibody,” refers to antibodies that are prepared,expressed, created or isolated by recombinant means, such as (a)antibodies isolated from an animal (e.g., a mouse) that is transgenic ortranschromosomal for immunoglobulin genes (e.g., human immunoglobulingenes) or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialantibody library (e.g., containing human antibody sequences) using phagedisplay, and (d) antibodies prepared, expressed, created or isolated byany other means that involve splicing of immunoglobulin gene sequences(e.g., human immunoglobulin genes) to other DNA sequences. Suchrecombinant antibodies may have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies can be subjected to in vitromutagenesis and thus the amino acid sequences of the V_(H) and V_(L)regions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline V_(H) and V_(L) sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species.

The term “human antibody,” as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences asdescribed, for example, by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies may include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The human antibody can have at least one or more amino acids replacedwith an amino acid residue, e.g., an activity enhancing amino acidresidue that is not encoded by the human germline immunoglobulinsequence. Typically, the human antibody can have up to twenty positionsreplaced with amino acid residues that are not part of the humangermline immunoglobulin sequence. In a particular embodiment, thesereplacements are within the CDR regions as described in detail below.

The term “humanized antibody” refers to an antibody that includes atleast one humanized antibody chain (i.e., at least one humanized lightor heavy chain). The term “humanized antibody chain” (i.e., a “humanizedimmunoglobulin light chain”) refers to an antibody chain (i.e., a lightor heavy chain, respectively) having a variable region that includes avariable framework region substantially from a human antibody andcomplementarity determining regions (CDRs) (e.g., at least one CDR, twoCDRs, or three CDRs) substantially from a non-human antibody, andfurther includes constant regions (e.g., one constant region or portionthereof, in the case of a light chain, and preferably three constantregions in the case of a heavy chain).

“Isolated,” as used herein, is intended to refer to an antibody orcombination of two, three or four antibodies that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated composition of antibodies ca, cf, and ch, each of whichspecifically bind to EGFR, is substantially free of antibodies thatspecifically bind antigens other than EGFR). In addition, an isolatedantibody is typically substantially free of other cellular materialand/or chemicals. In one embodiment, a combination of “isolated”monoclonal antibodies having different EGFR binding specificities arecombined in a well-defined composition.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In someembodiments, a monoclonal antibody composition provided herein comprisesonly antibodies of the IgG1 isotype. In other embodiments, a monoclonalantibody composition provided herein comprises only antibodies of theIgG2 isotype. In other embodiments, a monoclonal antibody compositionprovided herein comprises antibodies of two or three different isotypes.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich an antibody binds. In various embodiments, an antigen is EGF. In aparticular embodiment, an antigen is human EGFR.

Accordingly, also encompassed by the present disclosure are combinationsof antibodies that bind to epitopes on EGFR which comprise all or aportion of the epitopes recognized by the particular antibodies of thecombinations described herein. In another embodiment, the antibodies areprovided that compete for binding to EGFR with the antibodies describedherein. Competing antibodies and antibodies that recognize the same oran overlapping epitope can be identified using routine techniques suchas an immunoassay, for example, by showing the ability of one antibodyto block the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competitive binding may be determined usingan assay such as described in the Examples below.

The terms “specific binding,” “specifically binds,” “selective binding,”and “selectively binds,” mean that an antibody exhibits appreciableaffinity for a particular antigen or epitope and, generally, does notexhibit significant cross-reactivity with other antigens and epitopes.“Appreciable” or preferred binding includes binding with a K_(D) of 10⁶,10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹ or better. The K_(D) of an antibody antigeninteraction (the affinity constant) indicates the concentration ofantibody at which 50% of antibody and antigen molecules are boundtogether. Thus, at a suitable fixed antigen concentration, 50% of ahigher (i.e., stronger) affinity antibody will bind antigen molecules ata lower antibody concentration than would be required to achieve thesame percent binding with a lower affinity antibody. Thus a lower K_(D)value indicates a higher (stronger) affinity. As used herein, “better”affinities are stronger affinities, and are of lower numeric value thantheir comparators, with a K_(D) of 10⁷M⁻¹ being of lower numeric valueand therefore representing a better affinity than a K_(D) of 10⁶M⁻¹.Affinities better (i.e., with a lower K_(D) value and thereforestronger) than 10⁷M⁻¹, preferably better than 10⁸M⁻¹, are generallypreferred. Values intermediate to those set forth herein are alsocontemplated, and a preferred binding affinity can be indicated as arange of affinities, for example preferred binding affinities foranti-EGFR antibodies disclosed herein are, 10⁶ to 10¹²M⁻¹, preferably10⁷ to 10¹²M⁻¹, more preferably 10⁸ to 10¹² M⁻¹. An antibody that “doesnot exhibit significant cross-reactivity” is one that will notappreciably bind to an off target antigen (e.g., a non-EGFR protein).For example, in one embodiment, an antibody that specifically binds toEGFR will exhibit at least a two, and preferably three, or four or moreorders of magnitude better binding affinity (i.e., binding exhibiting atwo, three, or four or more orders of magnitude lower K_(D) value) forEGFR than for ErbB molecules other than ErbB1 (EGFR) or for non-ErbBproteins or peptides. Specific or selective binding can be determinedaccording to any art-recognized means for determining such binding,including, for example, according to Scatchard analysis and/orcompetitive (competition) binding assays as described herein.

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction or the affinity of an antibody for an antigen. In oneembodiment, the antibody provided herein binds an antigen (e.g., EGFR)with an affinity (K_(D)) of 100 nM or better (i.e., or less) (e.g., 90nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nMor less), as measured using a surface plasmon resonance assay, a cellbinding assay, or an equilibrium dialysis assay. In a particularembodiment, an antibody binds EGFR with an affinity (as represented bydissociation constant K_(D)) of 8 nM or better (e.g., 7 nM, 6 nM, 5 nM,4 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM or lower), asmeasured by a surface plasmon resonance assay or a cell binding assay.In other embodiments, an antibody binds an antigen (e.g., EGFR) with anaffinity (K_(D)) of approximately less than 10⁻⁷ M, such asapproximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by surface plasmon resonance (SPR) technology in a BIACORE3000 instrument using recombinant EGFR as the analyte and the antibodyas the ligand, and binds to the predetermined antigen with an affinitythat is at least two-fold greater than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen. Other methods for determiningK_(D) include equilibrium binding to live cells expressing EGFR via flowcytometry (FACS) or in solution using KinExA® technology.

The term “K_(off),” as used herein, is intended to refer to the off rateconstant for the dissociation of an antibody from the antibody/antigencomplex.

The terms “IC50” and “IC90,” as used herein, refer to the measure of theeffectiveness of a compound (e.g., an anti-EGFR antibody) in inhibitinga biological or biochemical function (e.g., the function or activity ofEGFR) by 50% and 90%, respectively. For example, IC50 indicates how muchof an anti-EGFR antibody is needed to inhibit the activity of EGFR(e.g., the growth of a cell expressing EGFR) by half. That is, it is thehalf maximal (50%) inhibitory concentration (IC) of an anti-EGFRantibody (50% IC, or IC₅₀). According to the FDA, IC50 represents theconcentration of a drug that is required for 50% inhibition in vitro.The IC50 and IC90 can be determined by techniques known in the art, forexample, by constructing a dose-response curve and examining the effectof different concentrations of the antagonist (i.e., the anti-EGFRantibody) on reversing EGFR activity.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody fragments (e.g., V_(H),V_(L), CDR3), is intended to refer to a nucleic acid molecule in whichthe nucleotide sequences are essentially free of other genomicnucleotide sequences, e.g., those encoding antibodies that bind antigensother than EGFR, which other sequences may naturally flank the nucleicacid in human genomic DNA.

The term “modifying,” or “modification,” as used herein, is intended torefer to changing one or more amino acids in the antibodies orantigen-binding portions thereof. The change can be produced by adding,substituting or deleting an amino acid at one or more positions. Thechange can be produced using known techniques, such as PCR mutagenesis.For example, in some embodiments, an antibody or an antigen-bindingportion thereof identified using the methods provided herein can bemodified, to thereby modify the binding affinity of the antibody orantigen-binding portion thereof to EGFR.

“Conservative amino acid substitutions” in the sequences of theantibodies are provided, i.e., nucleotide and amino acid sequencemodifications which do not abrogate the binding of the antibody encodedby the nucleotide sequence or containing the amino acid sequence, to theantigen, i.e., EGFR. Conservative amino acid substitutions include thesubstitution of an amino acid in one class by an amino acid of the sameclass, where a class is defined by common physicochemical amino acidside chain properties and high substitution frequencies in homologousproteins found in nature, as determined, for example, by a standardDayhoff frequency exchange matrix or BLOSUM matrix. Six general classesof amino acid side chains have been categorized and include: Class I(Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln,Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and ClassVI (Phe, Tyr, Trp). For example, substitution of an Asp for anotherclass III residue such as Asn, Gln, or Glu, is a conservativesubstitution. Thus, a predicted nonessential amino acid residue in ananti-EGFR antibody is preferably replaced with another amino acidresidue from the same class. Methods of identifying nucleotide and aminoacid conservative substitutions which do not eliminate antigen bindingare well-known in the art.

The term “non-conservative amino acid substitution” refers to thesubstitution of an amino acid in one class with an amino acid fromanother class; for example, substitution of an Ala, a class II residue,with a class III residue such as Asp, Asn, Glu, or Gln.

Alternatively, in another embodiment, mutations (conservative ornon-conservative) can be introduced randomly along all or part of ananti-EGFR antibody coding sequence, such as by saturation mutagenesis,and the resulting modified anti-EGFR antibodies can be screened forbinding activity.

A “consensus sequence” is a sequence formed from the most frequentlyoccurring amino acids (or nucleotides) in a family of related sequences.In a family of proteins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence. A “consensus framework” of animmunoglobulin refers to a framework region in the consensusimmunoglobulin sequence. Similarly, the consensus sequence for the CDRsof can be derived by optimal alignment of the CDR amino acid sequencesof EGFR antibodies provided herein.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art.

The nucleic acid compositions, while often comprising a native sequence(except for modified restriction sites and the like), from either cDNA,genomic or mixtures thereof may alternately be mutated, in accordancewith standard techniques to provide altered gene sequences. For codingsequences, these mutations, may modify the encoded amino acid sequenceas desired. In particular, DNA sequences substantially homologous tonative V, D, J, constant, switches and other such sequences describedherein are contemplated.

The term “operably linked” refers to a nucleic acid sequence placed intoa functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a pre-protein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice. A nucleicacid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms, “plasmid” and “vector” may be usedinterchangeably. However, other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions are alsocontemplated.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, an antibody or antibodypair or trio disclosed herein, for example, a subject having a diseaseor disorder associated with EGFR dependent signaling or predisposed tohaving such a disease or disorder, in order to prevent, cure, delay,reduce the severity of, or ameliorate one or more symptoms of thedisease or disorder or recurring disease or disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

As used herein, adjunctive or combined administration (coadministration)includes simultaneous administration of two or more compounds in thesame or different dosage form, or separate administration of two or morecompounds (e.g., sequential administration). For example, two or more ofthe antibodies can be simultaneously administered together (e.g.,formulated together). Alternatively, the antibodies can be administeredin combination (e.g., formulated for separate administration andadministered concurrently or sequentially). For example, a firstantibody can be administered followed by the administration of a secondantibody, or vice versa. Such concurrent or sequential administrationpreferably results in the two or more compounds (e.g., antibodies) beingsimultaneously present in treated patients.

The term “disease associated with EGFR dependent signaling,” or“disorder associated with EGFR dependent signaling,” as used herein,includes disease states and/or symptoms associated with a disease state,where increased levels of EGFR and/or activation of cellular cascadesinvolving EGFR are found. The term “disease associated with EGFRdependent signaling,” also includes disease states and/or symptomsassociated with the activation of alternative EGFR signaling pathways.In general, the term “disease associated with EGFR dependent signaling,”refers to any disorder, the onset, progression or the persistence of thesymptoms of which requires the participation of EGFR. ExemplaryEGFR-mediated disorders include, but are not limited to, for example,cancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastric cancer,pancreatic cancer, glial cell tumors such as glioblastoma andneurofibromatosis, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, melanoma,colorectal cancer, endometrial carcinoma, salivary gland carcinoma,kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer. Ina particular embodiment, a cancer treated or diagnosed using the methodsdisclosed herein is selected from melanoma, breast cancer, ovariancancer, renal carcinoma, gastrointestinal/colon cancer, lung cancer, andprostate cancer.

The term “effective amount,” as used herein, refers to that amount of anantibody or an antigen binding portion thereof that binds EGFR, which issufficient to effect treatment, prognosis or diagnosis of a diseaseassociated with EGFR dependent signaling, as described herein, whenadministered to a subject. Therapeutically effective amounts ofantibodies provided herein, when used alone or in combination, will varydepending upon the relative activity of the antibodies and combinations(e.g., in inhibiting cell growth) and depending upon the subject anddisease condition being treated, the weight and age of the subject, theseverity of the disease condition, the manner of administration and thelike, which can readily be determined by one of ordinary skill in theart. The dosages for administration can range from, for example, about 1ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng toabout 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg,about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ngto about 4,000 mg, about 1 μg to about 3,500 mg, about 5 μg to about3,000 mg, about 10 μg to about 2,600 mg, about 20 μg to about 2,575 mg,about 30 μg to about 2,550 mg, about 40 μg to about 2,500 mg, about 50μg to about 2,475 mg, about 100 μg to about 2,450 mg, about 200 μg toabout 2,425 mg, about 300 μg to about 2,000, about 400 μg to about 1,175mg, about 500 μg to about 1,150 mg, about 0.5 mg to about 1,125 mg,about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg toabout 1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950mg, about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg, about 5mg to about 875 mg, about 10 mg to about 850 mg, about 20 mg to about825 mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg toabout 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650mg, about 500 mg, or about 525 mg to about 625 mg, of an antibody.Dosage regiments may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (i.e., side effects) of an antibody are minimizedand/or outweighed by the beneficial effects.

The term “therapeutic agent” in intended to encompass any and allcompounds that have an ability to decrease or inhibit the severity ofthe symptoms of a disease or disorder, or increase the frequency and/orduration of symptom-free or symptom-reduced periods in a disease ordisorder, or inhibit or prevent impairment or disability due to adisease or disorder affliction, or inhibit or delay progression of adisease or disorder, or inhibit or delay onset of a disease or disorder,or inhibit or prevent infection in an infectious disease or disorder.Non-limiting examples of therapeutic agents include small organicmolecules, monoclonal antibodies, bispecific antibodies, recombinantlyengineered biologics, RNAi compounds, tyrosine kinase inhibitors, andcommercial antibodies. In certain embodiments, tyrosine kinaseinhibitors include, e.g., one or more of erlotinib, gefitinib, andlapatinib, which are currently marketed pharmaceuticals. Commerciallyavailable pharmaceutical anti-EGFR antibodies include cetuximab andpanitumumab. Other pharmaceutical anti-EGFR antibodies includezalutumumab, nimotuzumab, and matuzumab, which are in development.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

The term “subject” includes any mammal, e.g, a primate. For example, themethods and compositions herein disclosed can be used to treat a subjecthaving cancer. In a particular embodiment, the subject is a human.

The term “sample” refers to tissue, body fluid, or a cell (or a fractionof any of the foregoing) taken from a patient or a subject. Normally,the tissue or cell will be removed from the patient, but in vivodiagnosis is also contemplated. In the case of a solid tumor, a tissuesample can be taken from a surgically removed tumor and prepared fortesting by conventional techniques. In the case of lymphomas andleukemias, lymphocytes, leukemic cells, or lymph tissues can be obtained(e.g., leukemic cells from blood) and appropriately prepared. Othersamples, including urine, tears, serum, plasma, cerebrospinal fluid,feces, sputum, cell extracts etc. can also be useful for particularcancers.

The term “SN-38,” as used herein, refers to7-Ethyl-10-hydroxy-camptothecin, which is an active metabolite ofirinotecan commonly used in the art as a tool compound for irinotecan inin vitro experiments (CAS #86639-52-3). Irinotecan is a member of thetopoisomerase I inhibitor class of drugs and is a semi-synthetic andwater soluble analog of the naturally-occurring alkaloid, camptothecin.Also known as CPT-11, irinotecan is currently marketed formulated as anaqueous solution as Camptosar® (irinotecan hydrochloride injection).Topoisomerase I inhibitors such as irinotecan work to arrestuncontrolled cell growth by inhibiting the unwinding of DNA and therebypreventing DNA replication.

The pharmacology of irinotecan is complex, with extensive metabolicconversions involved in the activation, inactivation, and elimination ofthe drug. Irinotecan is a prodrug that is converted by nonspecificcarboxylesterases into a 100-1000 fold more active metabolite, SN-38.Because these esterases, which act on irinotecan within the body are notexpressed by many cells, including many, if not all, of the experimentalcell lines used in the Examples below, SN-38 is often substituted foririnotecan in cell culture experiments. Irinotecan hydrochlorideinjection is approved in the United States for treatment of metastaticcolon or renal cancer and is also used to treat colorectal, gastric,lung, uterine cervical and ovarian cancers.

Various aspects of the disclosure are described in further detail in thefollowing subsections.

II. Anti-EGFR Antibodies and Combinations Thereof

Novel anti-EGFR monoclonal antibodies are disclosed herein, includingthree referred to in the Examples as P1X, P2X and P3X. The P1X, P2X andP3X monoclonal antibodies are affinity matured antibodies of parentalantibodies referred to as ca, cd and ch, respectively, disclosed in PCTApplication No. PCT/US2011/35238. The CDR amino acid sequences of theparental and affinity matured antibodies are shown below, with thechanged amino acids in the affinity matured antibodies bolded andunderlined:

CDR1 CDR2 CDR3 ca V_(H) SYAIS IIPIFGTANY DPSVDL (SEQ ID NO: 1)(SEQ ID NO: 27) (SEQ ID NO: 28) P1X V_(H) SYAIS IIPIFGT V NY DPSV N L(SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) ca V_(L) QSISSWLA DASSLQQFAAHA (SEQ ID NO: 29) (SEQ ID NO: 5) (SEQ ID NO: 30) P1X V_(L) QSISSWW A DASSL QQ YH AH P (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6)cd V_(H) SYAIS IIPIFGTANY MGRGKV (SEQ ID NO: 7) (SEQ ID NO: 27)(SEQ ID NO: 9) P2X V_(H) SYAIS IIPIFG A AN P MGRGKV (SEQ ID NO: 7)(SEQ ID NO: 8) (SEQ ID NO: 9) cd V_(L) QSVLYSSNNKNYLA WASTR QQYYGSP(SEQ ID NO: 31) (SEQ ID NO: 11) (SEQ ID NO: 12) P2X V_(L) QSVLYS PNNKNYLA WASTR QQYYGSP (SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12)ch V_(H) SYGIN ISAYNGNTNY DLGGYGSGS (SEQ ID NO: 13) (SEQ ID NO: 32)(SEQ ID NO: 15) P3X V_(H) SYGIN ISAYNGNT Y Y DLGGYGSGS (SEQ ID NO: 13)(SEQ ID NO: 14) (SEQ ID NO: 15) ch V_(L) QSVSSNLA GASTR QDYRTWPR(SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) P3X V_(L) QSVSSNLA GASTRQDYRTWPR (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18)

The full-length V_(H) and V_(L) amino sequences for P1X are shown in SEQID NO: 19 and 20, respectively. The full-length V_(H) and V_(L) aminosequences for P2X are shown in SEQ ID NO: 21 and 22, respectively. Thefull-length V_(H) and V_(L) amino sequences for P3X are shown in SEQ IDNO: 23 and 24, respectively. Additionally, the V_(H) and V_(L) CDRsegments as presented herein are arranged, e.g., in the amino to carboxyterminal order of CDR1, CDR2 and CDR3.

In one embodiment, a monoclonal antibody is provided which binds EGFRextracellular domain and comprises heavy and light chain CDR1, CDR2, andCDR3, sequences, wherein the heavy and light chain CDR1, CDR2, and CDR3,sequences are selected from the group consisting of:

(a) heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and3 respectively, and light chain CDR1, CDR2, and CDR3 sequences of SEQ IDNOs: 4, 5, and 6, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 7, 8, and9, respectively, and light chain CDR1, CDR2, and CDR3 sequences of SEQID NOs: 10, 11 and 12, respectively; and

(c) heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 13, 14,and 15, respectively, and light chain CDR1, CDR2, and CDR3 sequences ofSEQ ID NOs: 16, 17, and 18, respectively.

In another embodiment, a monoclonal antibody is provided that binds toEGFR extracellular domain and comprises a heavy chain variable regionand a light chain variable region, wherein the heavy and light chainvariable region sequences are selected from the group consisting of:

(a) a heavy chain variable region comprising SEQ ID NO: 19 and a lightchain variable region comprising SEQ ID NO: 20;

(b) a heavy chain variable region comprising SEQ ID NO: 21 and a lightchain variable region comprising SEQ ID NO: 22; and

(c) a heavy chain variable region comprising SEQ ID NO: 23 and a lightchain variable region comprising SEQ ID NO: 24.

Combinations of the aforementioned anti-EGFR antibodies are alsoprovided. Such combinations can contain, for example, any combination oftwo of the aforementioned anti-EGFR antibodies. Another combination cancomprise all three of the aforementioned anti-EGFR antibodies.Accordingly, in another aspect, a composition comprising two or threemonoclonal antibodies which bind to EGFR extracellular domain isprovided, wherein the two or three monoclonal antibodies are selectedfrom the group consisting of:

(a) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;

(b) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively and

(c) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 13, 14, and 15, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17 and 18,respectively;

and wherein the composition comprises (a) and (b), (a) and (c), (b) and(c) or (a), (b) and (c).

In yet another embodiment, a composition comprising two or threemonoclonal antibodies which bind to EGFR extracellular domain isprovided, wherein the two or three monoclonal antibodies are selectedfrom the group consisting of:

(a) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 19 and a light chain variable region comprisingSEQ ID NO: 20;

(b) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 21 and a light chain variable region comprisingSEQ ID NO: 22; and

(c) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24; and wherein the composition comprises (a) and (b), (a)and (c), (b) and (c) or (a) (b) and (c).

The anti-EGFR antibodies disclosed herein, whether as a single antibodyor in a combination of antibodies, can bind to EGFR with a K_(D) of, forexample, better than 100 nM, or better than 10 nM or better than 1 nM.As used herein with respect to K_(D), the term “better than _(——————)nM” means that an antibody has a K_(D), expressed as a nanomolarconcentration, that is lower than the indicated number. For example, aK_(D) that is “better than” 100 nM indicates a K_(D), expressed as ananomolar concentration, that is lower in value than 100 nM (e.g., is 50nM).

In other embodiments, a P1X-related antibody, such as an antibodycomprising heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1,2, and 3 respectively, and light chain CDR1, CDR2, and CDR3 sequences ofSEQ ID NOs: 4, 5, and 6, respectively, or an antibody comprising V_(H)and V_(L) sequences of SEQ ID NOs: 19 and 20, respectively, can bind toEGFR with a K_(D) in a range of about 1×10⁻⁹ M to 1.1×10⁻¹¹ M or better.

In other embodiments, a P2X-related antibody, such as an antibodycomprising heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 7,8 and 9 respectively, and light chain CDR1, CDR2, and CDR3 sequences ofSEQ ID NOs: 10, 11, and 12, respectively, or an antibody comprisingV_(H) and V_(L) sequences of SEQ ID NOs: 21 and 22, respectively, canbind to EGFR with a K_(D) in a range of about 1×10⁻⁹ M to 7.0×10⁻¹¹ M orbetter.

In other embodiments, a P3X-related antibody, such as an antibodycomprising heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 13,14 and 15 respectively, and light chain CDR1, CDR2, and CDR3 sequencesof SEQ ID NOs: 16, 17, and 18, respectively, or an antibody comprisingV_(H) and V_(L) sequences of SEQ ID NOs: 23 and 24, respectively, canbind to EGFR with a K_(D) in a range of about 1×10⁻⁹ M to 3.6×10⁻¹⁰ M orbetter.

The anti-EGFR antibodies disclosed herein, whether as a single antibodyor in a combination of antibodies, can exhibit one or more otherfunctional properties as disclosed herein, including but not limited to

(a) inhibition of AKT or ERK phosphorylation, e.g., EGFR-dependant AKTor ERK phosphorylation, as measured in a cell-based assay;

(b) inhibition of the growth of cells expressing EGFR;

(c) inhibition of EGF ligand binding to EGFR (e.g., inhibition ofbinding of one or more ligands that bind EGFR, including EGF, heparinbinding EGF-like growth factor (HB-EGF), transforming growth factor(TGF), epigen, epiregulin, betacellulin, or amphiregulin);

(d) inhibition of EGFR dimerization;

(e) downregulation of EGFR on cell surfaces (e.g., by internalizationand recycling of the receptor, and/or internalization and degradation ofthe receptor);

(f) inhibition of in vitro tumor cell proliferation; and/or

(g) inhibition of in vivo tumor growth.

Antibodies disclosed herein include all known forms of antibodies andother protein scaffolds with antibody-like properties. For example, theantibody can be a human antibody, a humanized antibody, a bispecificantibody, an immunoconjugate, a chimeric antibody or a protein scaffoldwith antibody-like properties, such as fibronectin or ankyrin repeats.The antibody also can be a Fab, Fab′2, ScFv, Affibody®, avimer,nanobody, or a domain antibody. The antibody also can have any isotype,including any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM,IgA1, IgA2, IgAsec, IgD, and IgE. IgG antibodies are preferred.Full-length antibodies can be prepared from V_(H) and V_(L) sequencesusing standard recombinant DNA techniques and nucleic acid encoding thedesired constant region sequences to be operatively linked to thevariable region sequences. Non-limiting examples of suitable constantregion sequences include the kappa light chain constant region disclosedin SEQ ID NO: 25 and the IgG1 heavy chain constant region disclosed inSEQ ID NO: 26.

As disclosed in the examples, it has been discovered that triplecombinations of the P1X+P2X+P3X antibodies are particularly efficaciouswhen used at a P1X:P2X:P3X molar ratio of 2:2:1. Thus, for such a tripleantibody combination, 40% of the total concentration is selected to beP1X, 40% of the total concentration is selected to be P2X and 20% of thetotal concentration is selected to be P3X.

Accordingly, in another embodiment, is a composition is providedcomprising three monoclonal anti-EGFR antibodies, said compositioncomprising a first antibody, a second antibody and a third antibody,wherein (i) the first antibody comprises heavy chain CDR1, CDR2, andCDR3 sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;(ii) the second antibody comprises heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively; and (iii) the third antibody comprises heavy chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 13, 14, and 15 respectively, andlight chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17, and18, respectively, and wherein the first second and third antibodies arepresent at a molar ratio of 2:2:1 to each other.

In yet another embodiment, a composition is provided comprising threemonoclonal anti-EGFR antibodies, said composition comprising a firstantibody, a second antibody and a third antibody, wherein (i) the firstantibody comprises a heavy chain variable region comprising SEQ ID NO:19 and a light chain variable region comprising SEQ ID NO: 20; (ii) thesecond antibody comprises a heavy chain variable region comprising SEQID NO: 21 and a light chain variable region comprising SEQ ID NO: 22;and (iii) the third antibody comprises a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24, and wherein the first second and third antibodies arepresent at a molar ratio of 2:2:1 to each other.

Methods of preparing anti-EGFR antibody compositions comprising threeantibodies are provided, wherein the antibodies are prepared at a 2:2:1ratio. More specifically, in another embodiment, a method of preparingan anti-EGFR antibody composition is provided, the method comprisingcombining in a single composition:

(a) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;

(b) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively; and

(c) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 13, 14, and 15 respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17, and 18,respectively;

wherein (a), (b) and (c) are combined at a molar ratio of 2:2:1 to eachother.

In another embodiment, a method of preparing an anti-EGFR antibodycomposition is provided, the method comprising combining in a singlecomposition:

(a) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 19 and a light chain variable region comprisingSEQ ID NO:20;

(b) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 21 and a light chain variable region comprisingSEQ ID NO: 22; and

(c) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24;

wherein (a), (b) and (c) are combined at a molar ratio of 2:2:1 to eachother.

Methods of treating a subject with three anti-EGFR antibodies whereinthe antibodies are administered to the subject at a 2:2:1 ratio are alsoprovided. More specifically, a method of treating a subject withanti-EGFR antibodies is provided, the method comprising administering tothe subject:

(a) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;

(b) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively; and

(c) a monoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 13, 14, and 15 respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17, and 18,respectively;

wherein (a), (b) and (c) are administered to the subject at a molarratio of 2:2:1 to each other.

In another embodiment, a method of treating a subject with anti-EGFRantibodies is provided, the method comprising administering to thesubject:

(a) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 19 and a light chain variable region comprisingSEQ ID NO:20;

(b) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 21 and a light chain variable region comprisingSEQ ID NO: 22; and

(c) a monoclonal antibody comprising a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO: 24;

wherein (a), (b) and (c) are administered to the subject at a molarratio of 2:2:1 to each other.

Further details on formulating anti-EGFR antibodies into pharmaceuticalcompositions and methods of using such compositions in EGFR-relateddiseases are described in subsections below.

III. Methods for Producing Antibodies

(i) Monoclonal Antibodies

The monoclonal antibodies of provided herein most typically are preparedby standard recombinant DNA techniques based on the amino acid sequencesof the V_(H) and V_(L) regions disclosed herein.

Additionally or alternatively, monoclonal antibodies can be producedusing a variety of known techniques, such as the standard somatic cellhybridization technique, viral or oncogenic transformation of Blymphocytes, or yeast or phage display techniques using libraries ofhuman antibody genes. In particular embodiments, the antibodies arefully human monoclonal antibodies.

Accordingly, in one embodiment, a hybridoma method is used for producingan antibody that binds EGFR. In this method, a mouse or otherappropriate host animal can be immunized with a suitable antigen inorder to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the antigen used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes can then be fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. Culturemedium in which hybridoma cells are growing is assayed for production ofmonoclonal antibodies directed against the antigen. After hybridomacells are identified that produce antibodies of the desired specificity,affinity, and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods. Suitable culturemedia for this purpose include, for example, D-MEM or RPMI-1640 medium.In addition, the hybridoma cells may be grown in vivo as ascites tumorsin an animal. The monoclonal antibodies secreted by the subclones can beseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In another embodiment, antibodies that bind EGFR can be isolated fromantibody libraries generated using well know techniques such as thosedescribed in, for example, U.S. Pat. Nos. 5,223,409; 5,403,484; and5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 toDower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al. Additionally, production ofhigh affinity (nM range) human antibodies by chain shuffling, as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries may also be used. See, e.g.,U.S. patent application Ser. No. 09/856,907 (PCT Int. Pub. No. WO00/31246)

In a particular embodiment, the monoclonal antibody that binds EGFR isproduced using phage display. This technique involves the generation ofa human Fab library having a unique combination of immunoglobulinsequences isolated from human donors and having synthetic diversity inthe heavy-chain CDRs is generated. The library is then screened for Fabsthat bind to EGFR.

In yet another embodiment, human monoclonal antibodies directed againstEGFR can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system (see e.g.,U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all toLonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.).

In another embodiment, human antibodies can be raised using a mouse thatcarries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome (see e.g., PCTPublication WO 02/43478 to Ishida et al.).

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-EGFR antibodies. For example, an alternative transgenic systemreferred to as the Xenomouse (Abgenix, Inc.) can be used; such mice aredescribed in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-EGFR antibodies. For example, mice carrying both a human heavychain transchromosome and a human light chain tranchromosome can beused. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art and can be used to raiseanti-EGFR antibodies.

In yet another embodiment, antibodies can be prepared using a transgenicplant and/or cultured plant cells (such as, for example, tobacco, maizeand duckweed) that produce such antibodies. For example, transgenictobacco leaves expressing antibodies can be used to produce suchantibodies by, for example, using an inducible promoter. Also,transgenic maize can be used to express such antibodies and antigenbinding portions thereof. Antibodies can also be produced in largeamounts from transgenic plant seeds including antibody portions, such assingle chain antibodies (scFv's), for example, using tobacco seeds andpotato tubers.

The binding specificity of monoclonal antibodies (or portions thereof)that bind EGFR prepared using any technique including those disclosedhere, can be determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbentassay (ELISA). The binding affinity of a monoclonal antibody or portionthereof also can be determined by Scatchard analysis.

In certain embodiments, an EGFR antibody produced using any of themethods discussed above may be further altered or optimized to achieve adesired binding specificity and/or affinity using art recognizedtechniques, such as those described herein.

In one embodiment, partial antibody sequences derived from an EGFRantibody may be used to produce structurally and functionally relatedantibodies. For example, antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties. Such framework sequences can be obtained frompublic DNA databases that include germline antibody gene sequences.

Thus, one or more structural features of an anti-EGFR antibody, such asthe CDRs, can be used to create structurally related anti-EGFRantibodies that retain at least one desired functional property, e.g.,inhibiting growth of cells expressing EGFR.

In a particular embodiment, one or more CDR regions disclosed herein iscombined recombinantly with known human framework regions and CDRs tocreate additional, recombinantly-engineered, anti-EGFR antibodies. Theheavy and light chain variable framework regions can be derived from thesame or different antibody sequences.

It is well known in the art that antibody heavy and light chain CDR3domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen. Accordingly, incertain embodiments, antibodies are generated that include the heavyand/or light chain CDR3s of the particular antibodies described herein.The antibodies can further include the heavy and/or light chain CDR1and/or CDR2s of the antibodies disclosed herein.

The CDR 1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those disclosedherein. However, the ordinarily skilled artisan will appreciate thatsome deviation from the exact CDR sequences may be possible,particularly for CDR1 and CDR2 sequences, which can tolerate morevariation than CDR3 sequences without altering epitope specificity (suchdeviations are, e.g., conservative amino acid substitutions).Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDR1s and CDR2s that are, for example, 90%, 95%,98%, 99% or 99.5% identical to the corresponding CDRs of an antibodynamed herein.

In another embodiment, one or more residues of a CDR may be altered tomodify binding to achieve a more favored on-rate of binding. Using thisstrategy, an antibody having ultra high binding affinity of, forexample, 10¹⁰ M⁻¹ or more, can be achieved. Affinity maturationtechniques, well known in the art and those described herein, can beused to alter the CDR region(s) followed by screening of the resultantbinding molecules for the desired change in binding. Accordingly, asCDR(s) are altered, changes in binding affinity as well asimmunogenicity can be monitored and scored such that an antibodyoptimized for the best combined binding and low immunogenicity areachieved.

Modifications can also be made within one or more of the framework orjoining regions of the heavy and/or the light chain variable regions ofan antibody, so long as antigen binding affinity subsequent to thesemodifications is better than 10⁶ M⁻¹.

In another embodiment, the antibody is further modified with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers.Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.

Also provided are bispecific antibodies and immunoconjugates, asdiscussed below.

(ii) Bispecific Antibodies

Bispecific antibodies herein include at least two binding specificitiesfor EGFR which preferably bind non-overlapping or non-competingepitopes. Such bispecific antibodies can include additional bindingspecificities, e.g., a third EGFR binding specificity and/or a bindingspecificity for another ErbB receptor (e.g., ErbB3) or another antigen,such as the product of an oncogene. Bispecific antibodies can beprepared as full length antibodies or antibody fragments (e.g. F(ab′)₂bispecific antibodies).

Methods for making bispecific antibodies are well known in the art (see,e.g., WO 05117973 and WO 06091209). For example, production of fulllength bispecific antibodies can be based on the coexpression of twopaired immunoglobulin heavy chain-light chains, where the two chainshave different specificities. Various techniques for making andisolating bispecific antibody fragments directly from recombinant cellculture have also been described. For example, bispecific antibodieshave been produced using leucine zippers. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported.

In a particular embodiment, the bispecific antibody comprises a firstantibody (or binding portion thereof) which binds to EGFR derivatized orlinked to another functional molecule, e.g., another peptide or protein(e.g., another antibody or ligand for a receptor) to generate abispecific molecule that binds to at least two different binding sitesor target molecules. An antibody may be derivatized or linked to morethan one other functional molecule to generate multispecific moleculesthat bind to more than two different binding sites and/or targetmolecules; such multispecific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule, an antibody disclosed herein can be functionallylinked (e.g., by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, antibody fragment, peptide or binding mimetic, suchthat a bispecific molecule results.

Accordingly, bispecific molecules comprising at least one first bindingspecificity for EGFR and a second binding specificity for a secondtarget epitope are contemplated. In a particular embodiment, the secondtarget epitope is an Fc receptor, e.g., human FcγRI (CD64) or a humanFcα receptor (CD89). Therefore, bispecific molecules capable of bindingboth to FcγR, FcαR or FcεR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)), and to target cellsexpressing EGFR are also provided. These bispecific molecules targetEGFR expressing cells to effector cell and trigger Fc receptor-mediatedeffector cell activities, such as phagocytosis of an EGFR expressingcells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokinerelease, or generation of superoxide anion.

In one embodiment, the bispecific molecules comprise as a bindingspecificity at least one antibody, or an antibody fragment thereof,including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chain Fv. Theantibody may also be a light chain or heavy chain dimer, or any minimalfragment thereof such as a Fv or a single chain construct as describedin Ladner et al. U.S. Pat. No. 4,946,778.

The bispecific molecules can be prepared by conjugating the constituentbinding specificities, e.g., the anti-FcR and anti-EGFR bindingspecificities, using methods known in the art. For example, each bindingspecificity of the bispecific molecule can be generated separately andthen conjugated to one another. When the binding specificities areproteins or peptides, a variety of coupling or cross-linking agents canbe used for covalent conjugation. Examples of cross-linking agentsinclude protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate(SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),o-phenylenedimaleimide (oPDM),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC). Preferred conjugating agents are SATA and sulfo-SMCC, bothavailable from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule can bea single chain molecule comprising one single chain antibody and abinding determinant, or a single chain bispecific molecule comprisingtwo binding determinants. Bispecific molecules may comprise at least twosingle chain molecules. Methods for preparing bispecific molecules aredescribed for example in U.S. Pat. No. 5,260,203; U.S. Pat. No.5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat.No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S.Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or western blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA). The radioactive isotopecan be detected by such means as the use of a α γ-βcounter or ascintillation counter or by autoradiography.

(iii) Immunoconjugates

Immunoconjugates provided herein can be formed by conjugating theantibodies described herein to another therapeutic agent. Suitableagents include, for example, a cytotoxic agent (e.g., a chemotherapeuticagent), a toxin (e.g. an enzymatically active toxin of bacterial,fungal, plant or animal origin, or fragments thereof), and/or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-EGFR antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y and ¹⁸⁶Re.

Immunoconjugates can be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters(such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (see, e.g., WO94/11026).

IV. Methods for Screening Antibodies

Subsequent to producing antibodies they can be screened for variousproperties, such as those described herein, using a variety of assaysthat are well known in the art.

In one embodiment, the antibodies are screened (e.g., by flow cytometryor ELISA) for binding to EGFR using, for example, purified EGFR and/orEGFR-expressing cells, such as A431 cells. The epitopes bound by theanti-EGFR antibodies can further be identified and compared, forexample, to identify non-competing antibodies (e.g., antibodies thatbind different epitopes), as well as antibodies which compete forbinding and/or bind the same or overlapping epitopes.

Competitive antibodies and non-competitive antibodies can be identifiedusing routine techniques. Such techniques include, for example, animmunoassay, which shows the ability of one antibody to block (or notblock) the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competitive binding is determined in an assayin which the immunoglobulin under test inhibits specific binding of areference antibody to a common antigen, such as EGFR. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay; solid phase directbiotin-avidin EIA; solid phase direct labeled assay, solid phase directlabeled sandwich assay; solid phase direct ¹²⁵I labeled RIA; solid phasedirect biotin-avidin EIA; and direct labeled RIA. The surface plasmonresonance technique set forth in the Materials and Methods of theExamples and in Example 2, below, can also be used advantageously forthis purpose. Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.

Other screening techniques for determining the epitope bound byantibodies disclosed herein include, for example, x-ray analysis ofcrystals of antigen:antibody complexes, which provides atomic resolutionof the epitope. Other methods monitor the binding of the antibody toantigen fragments or mutated variations of the antigen where loss ofbinding due to a modification of an amino acid residue within theantigen sequence is often considered an indication of an epitopecomponent. In addition, computational combinatorial methods for epitopemapping can also be used. These methods rely on the ability of theantibody of interest to affinity isolate specific short peptides fromcombinatorial phage display peptide libraries. The peptides are thenregarded as leads for the definition of the epitope corresponding to theantibody used to screen the peptide library. For epitope mapping,computational algorithms have also been developed which have been shownto map conformational discontinuous epitopes.

In another embodiment, the antibodies (e.g., non-competing antibodiesanti-EGFR antibodies) are screened for the ability to bind to epitopesexposed upon binding to ligand, e.g., EGF (i.e., do not inhibit thebinding of EGFR-binding ligands to EGFR). Such antibodies can beidentified by, for example, contacting cells which express EGFR (e.g.A431 cells) with a labeled EGFR ligand (e.g., radiolabeled orbiotinylated EGF) in the absence (control) or presence of the anti-EGFRantibody. If the antibody does not inhibit EGF binding to EGFR, then nostatistically significantly decrease in the amount of label recovered,relative to the amount in the absence of the antibody, will be observed.Alternatively, if the antibody inhibits EGF binding to EGFR, then astatistically significantly decrease in the amount of label recovered,relative to the amount in the absence of the antibody, will be observed.

Antibodies also can be screened (tested) for their binding affinity.This can be done, for example, using a plasmon resonance assay, e.g., asdescribed below.

Antibodies also can be screened for their ability to inhibit signalingthrough EGFR using signaling assays, such as, those described herein.For example, the ability of an antibody to inhibit EGFR ligand mediatedphosphorylation of EGFRs can be assessed by treating cells expressingEGFR with an EGFR ligand (e.g., EGF) in the presence and absence of theantibody. The cells can then be lysed, crude lysates centrifuged toremove insoluble material, and EGFR phosphorylation measured, forexample, by western blotting followed by probing with ananti-phosphotyrosine antibody.

Alternatively, the ability of an antibody to inhibit downstreamsignaling through EGFR can be measured by kinase assays for knownsubstrates of EGFR such as, for example, AKT and/or ERK, following EGFRstimulation by EGF ligand. For example, cells expressing EGFR can beincubated with a candidate antibody and stimulated with EGF ligand. Celllysates subsequently prepared from such cells can be immunoprecipitatedwith an antibody for a substrate of EGFR (or a protein in a cellularpathway involving EGFR) such as, an anti-AKT antibody, and assayed forkinase activity (e.g., AKT kinase activity) using art-recognizedtechniques. A decrease in or complete disappearance in level or activity(e.g., kinase activity) of a EGFR substrate or protein in a pathwayinvolving EGFR in the presence of the antibody, relative to the level oractivity in the absence of the antibody is indicative of an antibodywhich inhibits EGFR signaling.

Antibodies that decrease levels of EGFR on cell surfaces can beidentified by their ability to downregulate or inhibit EGFR expressionon tumor cells. In certain embodiments, the antibodies decrease EGFR oncell surfaces by inducing internalization (or increasing endocytosis) ofEGFR (e.g., by internalization and recycling of the receptor and/orinternalization and degradation of the receptor) or by inhibitingrecycling of internalized EGFR. To test this, EGFR can be biotinylatedand the number of EGFR molecules on the cell surface can be readilydetermined, for example, by measuring the amount of biotin on amonolayer of cells in culture in the presence or absence of an antibody,followed by immunoprecipitation of EGFR and probing with streptavidin. Adecrease in detection of biotinylated EGFR over time in the presence ofan antibody is indicative of an antibody that decreases EGFR levels oncell surfaces.

Antibodies and antibody combinations can also be tested for theirability to inhibit growth of cells expressing EGFR (either in vivo or invitro), such as tumor cells, using art recognized techniques, includingthe Cell Titer-Glo Assay described in the Examples below andTritium-labeled thymidine incorporation assay. Antibodies also can bescreened for the ability to inhibit spheroid growth of cells expressingEGFR. This can be done by using an assay that approximates conditions ofa developing tumor growth as described herein.

In another embodiment, combinations of anti-EGFR antibodies are screenedfor IC50 and/or IC90 values relative to inhibiting a particular EGFRactivity or function, such as EGFR-mediated signaling (e.g., as measuredby ELISA, Western, or multiplex methods, such as Luminex®). Combinationsof antibodies, each of which possesses a particularly desired IC50and/or IC90 value (e.g., an IC90 of about 80 nM for inhibiting EGFRsignaling) can then be selected. In one embodiment, the combination hasa greater IC50 or IC90 value than a known reference antibody (e.g.,cetuximab). In another embodiment, the combination has an additive IC50or IC90 (i.e., the sum of the activities of the antibodies, when actingindividually on a cell expressing EGFR, is approximately equivalent tothe combined effect of the same antibodies acting together on the samecell) In another embodiment, the combination has a synergistic IC50 orIC90 (i.e., the sum of the effects of the antibodies, when actingindividually on a cell expressing EGFR, is less than the combined effectof the same antibodies acting together on the same cell).

V. Pharmaceutical Compositions

In another aspect, herein provided is a composition, e.g., apharmaceutical composition, containing one or a combination of theanti-EGFR monoclonal antibodies disclosed herein, formulated togetherwith a pharmaceutically acceptable carrier. In one embodiment, thecompositions include a combination of multiple (e.g., two or three)isolated antibodies that bind different epitopes on EGFR. Suchantibodies preferably have an additive or synergistic effect relative toinhibiting a particular EGFR activity or function, such as EGFR-mediatedsignaling. Preferred pharmaceutical compositions are sterilecompositions, compositions suitable for injection, and sterilecompositions suitable for injection by a desired route ofadministration, such as by intravenous injection.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

Compositions can be administered alone or in combination therapy, i.e.,combined with other agents. For example, the combination therapy caninclude a composition provided herein with at least one or moreadditional therapeutic agents, such as the anti-cancer agents describedherein. In one embodiment, combination therapy can use a compositionprovided herein of two or three of the anti-EGFR antibodies disclosedherein. In another embodiment, combination therapy can use a compositioncomprising at least one of the anti-EGFR antibodies disclosed hereincombined with one or more other antibodies, such as one or more otheranti-EGFR antibodies known in the art (e.g., anti-EGFR antibodies asdisclosed in PCT Application No. PCT/US2011/3528). The compositions canalso be administered in conjunction with radiation therapy and/orsurgery. Particular combinations of anti-EGFR antibodies may also beadministered separately or sequentially, with or without additionaltherapeutic agents.

Compositions can be administered by a variety of methods known in theart. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results. Theantibodies can be prepared with carriers that will protect theantibodies against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art.

To administer compositions by certain routes of administration, it maybe necessary to coat the constituents, e.g., antibodies, with, orco-administer the compositions with, a material to prevent itsinactivation. For example, the compositions may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Acceptable diluents include saline and aqueous buffer solutions.Liposomes include water-in-oil-in-water CGF emulsions as well asconventional liposomes.

Acceptable carriers include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. Except insofaras any conventional medium or agent is incompatible with the antibodies,use thereof in compositions provided herein is contemplated.Supplementary active constituents can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Including inthe composition an agent that delays absorption, for example,monostearate salts and gelatin can bring about prolonged absorption ofthe injectable compositions.

Sterile injectable solutions can be prepared by incorporating themonoclonal antibodies in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by sterilization microfiltration. Generally, dispersions areprepared by incorporating the antibodies into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, human antibodiesmay be administered once or twice weekly by subcutaneous injection oronce or twice monthly by subcutaneous injection.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of antibodies calculated to producethe desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit formsprovided herein are dictated by and directly dependent on (a) the uniquecharacteristics of the antibodies and the particular therapeutic effectto be achieved, and (b) the limitations inherent in the art ofcompounding such antibodies for the treatment of sensitivity inindividuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations include those suitablefor oral, nasal, topical (including buccal and sublingual), rectal, andparenteral administration. Parenteral administration is the most commonroute of administration for therapeutic compositions comprisingantibodies. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods known in the art ofpharmacy. The amount of antibodies that can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Thisamount of antibodies will generally be an amount sufficient to produce atherapeutic effect. Generally, out of one hundred percent, this amountwill range from about 0.001 percent to about ninety percent of antibodyby mass, preferably from about 0.005 percent to about 70 percent, mostpreferably from about 0.01 percent to about 30 percent.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions provided herein include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Particularexamples of adjuvants which are well-known in the art include, forexample, inorganic adjuvants (such as aluminum salts, e.g., aluminumphosphate and aluminum hydroxide), organic adjuvants (e.g., squalene),oil-based adjuvants, virosomes (e.g., virosomes which contain amembrane-bound heagglutinin and neuraminidase derived from the influenzavirus).

Prevention of presence of microorganisms may be ensured both bysterilization procedures and by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, and the like. It may also be desirable to include isotonicagents, such as sugars, sodium chloride, and the like into thecompositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of one or moreagents that delay absorption such as aluminum monostearate or gelatin.

When compositions are administered as pharmaceuticals, to humans andanimals, they can be given alone or as a pharmaceutical compositioncontaining, for example, 0.001 to 90% (more preferably, 0.005 to 70%,such as 0.01 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

Regardless of the route of administration selected, compositionsprovided herein, may be used in a suitable hydrated form, and they maybe formulated into pharmaceutically acceptable dosage forms byconventional methods known to those of skill in the art.

Actual dosage levels of the antibodies in the pharmaceuticalcompositions provided herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions employed, or the ester, saltor amide thereof, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts. A physician or veterinarian having ordinary skill in theart can readily determine and prescribe the effective amount of thecomposition required. For example, the physician or veterinarian couldstart doses of the antibodies at levels lower than that required toachieve the desired therapeutic effect and gradually increasing thedosage until the desired effect is achieved. In general, a suitabledaily dose of compositions provided herein will be that amount of theantibodies which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for antibodies to be administered alone, it is preferable toadminister antibodies as a formulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art, such as, for example, those disclosed in U.S. Pat. Nos.5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,4,596,556, 4,487,603, 4,486,194, 4,447,233, 4,447,224, 4,439,196, and4,475,196.

In certain embodiments, the monoclonal antibodies can be formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure thattherapeutic antibodies cross the BBB (if desired), they can beformulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; 5,399,331;5,891,468; 6,056,973; 6,210,707, 6,224,903; 6,316,024; 7,122,202;7,135,177; and 7,507,407 and US Patent Publication 20070116753. Theliposomes may comprise one or more moieties that attach to and/or areselectively transported into specific cells or organs, thus enhancetargeted drug delivery.

Pharmaceutical compositions are provided that comprise trios ofanti-EGFR antibodies at a 2:2:1 ratio, that is the composition comprisesthree different anti-EGFR antibodies, in particular a P1X-relatedantibody, a P2X-related antibody and a P3X-related antibody, which bindto different EGFR epitopes, formulated at a specific 2:2:1 ratio. Inaddition to the three antibodies, these pharmaceutical compositions cancomprise a pharmaceutically acceptable carrier and/or other excipient(s)such as those described in detail above. The pharmaceutical compositioncan be supplied in a single container containing all three antibodiesor, alternatively, the pharmaceutical composition can comprise a packagecomprising three distinct containers each containing one of the threedifferent anti-EGFR antibodies (as well as a pharmaceutically acceptablecarrier and/or other excipient(s) as described above).

Uses of the above-described anti-EGFR antibodies are provided, eitheralone (as single agents), in pair combinations (two antibodies), or intriple combinations (three antibodies) in the manufacture of amedicament for the treatment of a disease associated with EGFR dependentsignaling. The above-described anti-EGFR antibodies are also provided,either alone (as single agents), in pair combinations (two antibodies),or in triple combinations (three antibodies) for the treatment of cancer(or to be used in the manufacture of a medicament for the treatment ofcancer), such as an EGFR-expressing cancer, such as a cancer including,but not limited to melanoma, breast cancer, ovarian cancer, renalcarcinoma, gastrointestinal cancer, colon cancer, lung cancer,pancreatic cancer, skin cancer, head and neck cancer glioblastoma,prostate cancer and other solid and/or metastatic tumors.

Additionally, contemplated compositions may further include, or beprepared for use as a medicament in combination therapy with, anadditional therapeutic agent, e.g., an additional anti-cancer agent. An“anti-cancer agent” is a drug used to treat tumors, cancers,malignancies, and the like. Drug therapy (e.g., with antibodycompositions disclosed herein) may be administered without othertreatment, or in combination with other treatments such as surgery,heat, or radiation therapy (e.g., with ionizing radiation). Severalclasses of anti-cancer agents may be used in cancer treatment, dependingon the nature of the organ or tissue involved. For example, breastcancers are commonly stimulated by estrogens, and may be treated withdrugs which inactive sex hormones. Similarly, prostate cancer may betreated with drugs that inactivate androgens. Anti-cancer agents for usein combination with antibody compositions disclosed herein include,among others, those listed in Appendix A, which should not be construedas limiting. One or more anti-cancer agents may be administered eithersimultaneously or before or after administration of an antibodycomposition disclosed herein. Antibody compositions disclosed herein canbe administered sequentially or together with the additional anti-canceragent, e.g., an anti cancer agent disclosed in Appendix A, below.

Also provided are kits comprising one or more anti-EGFR antibodiesdisclosed herein, optionally contained in a single vial or container,and include, e.g., instructions for use in treating or diagnosing adisease associated with EGFR upregulation and/or EGFR dependentsignaling (e.g., a cancer such as those described in subsection VIbelow). The kits may include a label indicating the intended use of thecontents of the kit. The term label includes any writing, marketingmaterials or recorded material supplied on or with the kit, or whichotherwise accompanies the kit. Such kits may comprise the antibodycomposition in unit dosage form, such as in a single dose vial or asingle dose pre-loaded syringe. Kits comprising a combination of theanti-EGFR antibodies disclosed herein (e.g., a combination of aP1X-related antibody, a P2X-related antibody, and a P3X-relatedantibody) can comprise a single vial containing all components of thecombination or, alternatively, the kit can comprise each component inseparate vials with instructions for administration of the antibodies incombination therapy. In a preferred embodiment, a P1X-related antibody,a P2X-related antibody, and a P3X-related antibody are supplied in a2:2:1 ratio in a single vial or, alternatively, are supplied each inseparate vials with instructions for administering the three antibodiesat a 2:2:1 ratio.

VI. Methods of Using Antibodies

Antibodies and compositions disclosed herein can be used in a broadvariety of therapeutic and diagnostic applications, particularlyoncological applications. Accordingly, in another aspect, providedherein are methods for inhibiting EGFR activity in a subject byadministering one or more antibodies or compositions described herein inan amount sufficient to inhibit EGFR-mediated activity. Particulartherapeutic indications which can be treated include, for example,cancers of organs or tissues such as skin, brain and central nervoussystem, head and neck, esophagus, stomach, colon, rectum, anus, liver,pancreas, bile duct, gallbladder, lung or bronchus, breast, ovary,uterus, cervix, vagina, testis, germ cells, prostate, kidney, ureter,urinary bladder, adrenal, pituitary, thyroid, bone, muscle or otherconnective tissues, leukemia, multiple myeloma, Hodgkin's lymphoma andnon-Hodgkin's lymphoma.

Antibodies of disclosed herein also can be used to diagnose or prognosediseases (e.g., cancers) associated with EGFR, for example, bycontacting one or more antibodies, antibody pairs or antibody triosdisclosed herein (e.g., ex vivo or in vivo) with cells from the subject,and measuring the level of binding to EGFR on the cells, whereinabnormally high levels of binding to EGFR indicate that the subject hasa cancer associated with EGFR.

Also provided are methods of using the anti-EGFR antibodies disclosedherein in a variety of ex vivo and in vivo diagnostic and therapeuticapplications involving EGFR dependent signaling, including a variety ofcancers.

Accordingly, in one embodiment, a method is provided for treating adisease associated with EGFR dependent signaling by administering to asubject an antibody or preferably a combination of antibodies providedherein in an amount effective to treat the disease. Suitable diseasesinclude, for example, a variety of cancers including, but not limitedto, melanoma, breast cancer, ovarian cancer, renal carcinoma,gastrointestinal cancer, colon cancer, lung cancer, pancreatic cancer,skin cancer, head and neck cancer glioblastoma, prostate cancer andother solid and/or metastatic tumors.

The antibody can be administered alone or with another therapeutic agentthat acts in conjunction with or synergistically with the antibody totreat the disease associated with EGFR mediated signaling. Suchtherapeutic agents include those described herein, for example, smallorganic molecules, monoclonal antibodies, bispecific antibodies,recombinantly engineered biologics, RNAi compounds, tyrosine kinaseinhibitors, and commercial antibodies, as well as anticancer agents(e.g., cytotoxins, chemotherapeutic agents, small molecules andradiation). Non-limiting examples of anti-cancer agents that can be usedin combination therapy with one or more of the anti-EGFR antibodiesdisclosed herein include, for example, the anti-ErbB3 antibody MM-121and irinotecan. Further non-limiting examples are listed in Appendix A.

In one embodiment, the antibody is coadmistered with another antibody oragent. Coadministration includes simultaneous administration of thecompounds in the same or different dosage form, or separateadministration of the compounds (e.g., sequential administration). Forexample, two or more of the antibodies can be simultaneouslyadministered together (e.g., formulated together). Alternatively, theantibodies can be administered in combination (e.g., formulatedseparately and administered concurrently or sequentially). For example,a first antibody can be administered followed by the administration of asecond antibody, or vice versa. Such concurrent or sequentialadministration preferably results in the two or more compounds orantibodies being simultaneously present in treated patients.

Other embodiments are described in the following non-limiting Examples.

EXAMPLES Materials and Methods for the Examples

In general, in the following examples, unless otherwise indicated,conventional techniques of chemistry, molecular biology, recombinant DNAtechnology, immunology (especially, e.g., antibody technology), andstandard techniques in recombinant immunoglobulin preparation were used.

Cell Lines

All the cell lines to be used in the experiments described below areobtained from the National Cancer Institute (NCI), Cell Lines Service(CLS), Sigma-Aldrich Co. LLC. (Sigma), or ATCC.

TABLE 0 Cell Lines: Cell Line Source Catalog # Type A431 ATCC ATCC ®human epidermoid carcinoma CRL-1555 ™ DU145 ATCC ATCC ® human prostatecarcinoma HTB-81 ™ H1975 ATCC ATCC ® human lung non-small cell lungCRL-5908 ™ adenocarcinoma HCC827 ATCC ATCC ® human lung non-small celllung CRL-2868 ™ adenocarcinoma A549 ATCC ATCC ® human lung carcinomaCCL-185 ™ BxPC-3 ATCC ATCC ® human pancreatic adenocarcinoma CRL-1687 ™NCI-H1355 ATCC ATCC ® human lung adenocarcinoma CRL-5865 ™ NCI-H226 ATCCATCC ® human lung squamous cell CRL-5826 ™ carcinoma/mesotheliomaNCI-H322M Sigma 95111734 human lung non-small cell lung adenocarcinomaNCI-H358 ATCC ATCC ® human lung bronchioalveolar CRL-5807 ™ carcinoma;non-small cell lung cancer HCT 116 ATCC ATCC ® human colon carcinomaHOP-62 NCI CCL-247 ™ human lung carcinoma HT-1197 ATCC ATCC ® humanbladder carcinoma CRL-1473 ™ HT-29 ATCC ATCC ® human colorectaladenocarcinoma HTB-38 ™ LoVo ATCC ATCC ® human colorectal adenocarcinomaCCL-229 ™ RT-112 CLS 300324 human bladder carcinoma SCaBER ATCC ATCC ®human bladder squamous cell HTB-3 ™ carcinoma SK-MES-1 ATCC ATCC ® humanlung squamous cell HTB-58 ™ carcinoma SW 900 ATCC ATCC ® human lungsquamous cell HTB-59 ™ carcinomaProtein Purification of EGFR Extracellular Domain (EGFR-ECD) Mutants

Mutants of the EGFR extracellular domain (EGFR-ECD) are generated formAb epitope binning. Mutations were designed based upon both thecetuximab (Li S. et al., Cancer Cell. 7: 301-311, 2005) and H11(Spangler J. et al. PNAS. 107: 13252-13257, 2010) epitopes and uponstructural analysis of the EGFR-ECD structure (Protein Data Bank ID:1NQL; Ferguson K. M. et al. Mol Cell. 11: 507-517, 2003). Residues aremutated to alanines as noted in the protein sequences included in thisapplication. DNA synthesis of expression constructs may be commerciallyobtained from DNA2.0 (www.dna20.com). Subsequent DNA subcloning, proteinexpression in 293F cells and protein purification are completed usingconventional methods.

Inhibition of EGF-Mediated Signaling of EGFR or ERK in Tumor Cells

Inhibition of ligand-mediated tumor cell signaling is investigated asfollows: A431 or Du145 cells are seeded at a density of 35,000cells/well or 17,500 cells per half well in 96 well tissue cultureplates and grown in DMEM or RPMI-1640 medium supplemented withantibiotics, 2 mM L-glutamine and 10% fetal bovine serum (FBS) for 24hours at 37° C. and 5% carbon dioxide. Cells are serum starved in 1% FBSmedium with antibiotics and 2 mM L-glutamine for about 20 hours at 37°C. and 5% carbon dioxide. Cells are then preincubated with varyingconcentrations of anti-EGFR antibodies for 2 hrs, and then stimulatedwith human EGF ligand (50 ng/ml) (PeproTech, cat # AF-100-15) for 10minutes at 37° C. and 5% carbon dioxide. Cells are washed with ice-coldPBS and lysed in 500 ice-cold Lysis buffer (Mammalian Protein ExtractLysis buffer (MPER-Pierce, #78505) amended with 150 mM NaCl and proteaseinhibitor cocktail (Sigma, P714)) by incubating on ice for 30 minutes.Lysates are either analyzed immediately by ELISA for ERK (a downstreameffector of EGFR) and EGFR phosphorylation, or frozen at −80° C. untiluse.

ELISA Assays

For the phospho-EGFR sandwich ELISA, 96-half well GREINER high bindingplates (Cat. #675077; GREINER BIO-ONE, Monroe, N.C.) are coated with 50μL of an EGFR antibody (EGFR Ab-11, Clone: 199.12, without BSA andazide, Fisher Scientific, cat# MS396P1ABX), and incubated overnight atroom temperature. The next morning, the plates are washed 3 times with100 μl/well PBST (0.05% Tween-20) on a BIOTEK plate washer. Plates aresubsequently blocked for about 1 hour at room temperature with 2% BSA inPBS. The plates are washed 3 times with 100 μl/well PBST (0.05%Tween-20) on the BIOTEK plate washer. Cell lysates (50 μl) or standards(pEGFR pY1068 ELISA kit, R&D Systems, cat# DYC3570) are diluted in 50%Lysis buffer and 1% BSA-PBS (per the manufacturer's recommendations) andare added to the plates in duplicates and incubated for 2 hrs at roomtemperature or overnight at 4° C. with shaking. Plates are then washed 3times with 100 μl/well in the BIOTEK plate washer with PBST (PBS with0.05% Tween-20). About 50 μl of a detection antibody conjugated to horseradish peroxidase (HRP) (pEGFR pY1068 ELISA kit, R&D Systems, cat#DYC3570) diluted in 2% BSA, PBS is added and incubated for about 2 hourat room temperature. The plate is washed 3 times with 100 μl/well in theBIOTEK plate washer with PBST (0.05% Tween-20). About 50 μL ofSUPERSIGNAL PICO ELISA substrate is added and the plate is read using anEnvision (Perkin Elmer) plate reader. For data analysis, duplicatesamples are averaged and error bars are used to represent the standarddeviation between the two replicates. Inhibition curves andcorresponding IC50 values are calculated using GraphPad Prism software(GraphPad Software, Inc.) via regression of the data to a 4 parameterlogistic equation.

The phospho-ERK ELISA is performed similarly to the phospho-EGFR ELISAwith the following changes: Human pERK ELISA Duoset kit is purchasedfrom R&D Systems (cat# DYC1018-5) and used as recommended by themanufacturer.

A direct ELISA is performed using EGFR-ECD wild-type (WT), a Bin1epitope mutant, or a Bin3 epitope mutant as capture reagents (4 μg/ml).96-half well GREINER high binding plates (Cat. #675077; GREINER BIO-ONE,Monroe, N.C.) are coated with 50 μL of capture reagent and incubatedovernight at room temperature. Next morning, plates are washed 3 timeswith 100 μl/well in a BIOTEK plate washer with PBST (0.05% Tween-20) andblocked for about 1 hour at room temperature with 1% BSA in PBS, pH7.2.Varying concentrations (1, 0.25, 0.06, and 0.02 μg/ml) of monoclonalantibodies (mAbs) diluted in 1% BSA in PBS, pH7.2 are incubated with thecapture reagents at room temperature for 2 hours, followed by detectionwith 1:50,000 dilution in 1% BSA in PBS, pH7.2 of Peroxidase-ConjugatedAffiniPure Goat Anti-Human IgG Fc Fragment (Jackson ImmunoresearchCatalog #109-035-008) for 2 hours. About 50 μL of Supersignal PICO ELISAsubstrate is added and the plate is read using an Envision (PerkinElmer) plate reader. For data analysis, duplicate samples are averagedand error bars are used to represent the standard deviation between thetwo replicates.

Binding Affinity: Kinetic Exclusion Assay (KinExA)

Affinities and cross reactivity of antibodies are measured in solutionwith recombinant EGF receptor using KinExA instrumentation (SAPIDYNEInstruments, Boise, Id.). Materials used for this assay are a KinExA3000 instrument and software (Sapidyne Instruments, Boise, Id.),polymethylmethacrylate (PMMA) beads (Sapidyne Instruments), humananti-EGFR IgG, recombinant human EGFR, Cy5-conjugated goat anti-humanIgG (Jackson ImmunoResearch, West Grove, Pa.), phosphate buffered saline(PBS), and bovine serum albumin in PBS (100 mg/ml).

To couple the recombinant EGF receptor to PMMA beads, 100 μg ofrecombinant EGFR is added to a pre-measured aliquot of 200 mg PMMAbeads, and PBS is added to make the total volume 1 ml. The beads areincubated for 1 hr at room temperature on a rotating wheel. Then thebeads are briefly centrifuged and the supernatant is removed. 100 μl of100 mg/ml BSA in PBS is added to the beads, with further addition of PBSto make a total volume of 1 ml. The beads are incubated again for 1 hrat room temperature on a rotating wheel. The beads are then transferredto a glass bottle containing 27 mL of PBS.

To determine the monovalent antibody binding affinity, a twelve-stepdilution series of recombinant EGFR (75 nM, 25 nM, 8.3 nM, 2.8 nM, 0.9nM, 0.3 nM, 100 pM, 33 pM, 11 pM, 4 pM, 1.3 pM, 0 pM) is prepared in 5ml PBS having a constant concentration of anti-EGFR antibody. Foraccurate affinity measurement, the total antibody binding siteconcentration (“ABC”; twice the molar concentration of antibody, due tovalence) should be less than the monovalent affinity of the antibody forEGFR. The antibody-receptor mixtures are incubated for 2 hr at roomtemperature in order to achieve equilibrium. Depending upon the expectedaffinity of the antibody-receptor complex, this equilibration time maybe adjusted accordingly. In a separate tube, 15 mL of 2 μg/mLCy5-conjugated anti-human IgG secondary antibody is prepared, using a1:1000 dilution of stock (2 mg/mL) antibody into PBS. Then, the KinExAinstrument lines are attached to each of the 12 antibody-receptorsolution tubes. Each solution is injected through a packed EGFR-beadcolumn. (The KinExA instrument automatically packs a fresh bead columnfor each injection.) After a wash step, the labeled secondary antibodyis passed through the column. Finally, using the measured amount ofuncomplexed receptor at different receptor concentrations, theequilibrium titration data is fit to a 1:1 binding model in the KinExAsoftware to yield an affinity value K_(D). The lower the value of K_(D)the better (stronger, sometimes stated as higher) the binding affinity.Therefore a recitation that an antibody binds with a K_(D) of x nM orbetter means it binds with a K_(D) value of, e.g., 1×10⁻⁸M (10 nM) orwith a lower K_(D) value, e.g., 1×10⁻¹⁰ M (0.1 nM), with the lower K_(D)value indicating better (higher) affinity.

To determine the binding on-rate using the KinExA “direct method”, theequilibrium monovalent binding affinity (K_(D)) is determined using theabove approach and total antibody binding site concentration (ABC).Then, using the “Theoretical Binding Curve Demonstration” software(Sapidyne Instruments), the starting antigen concentration (L0) isdetermined for the kinetics experiment. To do this, the affinity and ABCvalues determined in the monovalent binding affinity experiment areentered, and a starting antigen concentration is selected as thatconcentration where roughly 20% of antibody will be unbound to antigenat equilibrium. This assures good signal-to-noise ratio in theexperiment. 15 mL of 2 μg/mL Cy5-conjugated anti-human IgG secondaryantibody is prepared, using a 1:1000 dilution of stock (2 mg/mL)antibody into PBS. In a separate tube, 8 mL of anti-EGFR antibodysolution is prepared at a concentration of 2×ABC. This concentration isdouble the running concentration, since it is mixed with 8 mL of antigensolution prior to the experiment. In a separate tube, 8 mL ofrecombinant EGFR solution is prepared at a concentration of 2×L0. Thisconcentration is also double the running concentration, since it ismixed with 8 mL of antibody solution prior to the experiment. Then, theEGFR coated beads and secondary antibody solution are placed in theappropriate container and line, respectively. The antibody and antigensolution are mixed thoroughly and immediately connected to theappropriate line, and the KinExA software is used to measure the amountof free antibody as a function of time in the resulting solution. Todetermine the association constant k_(on) (Kon), the KinExA software isused to fit the depletion of the amount of free antibody as a functionof time to a reversible bimolecular rate equation. The dissociationconstant K_(off) (Koff) is equal to the K_(on)*K_(D) (Kd).

Binding Affinity: Surface Plasmon Resonance Assay

The Surface Plasmon Resonance Assay is performed as follows:

either antibody or antigen (300 RU) is immobilized on a CMS chip usingamine coupling. Different concentrations of antibodies or antigens arethen injected to study their association and dissociation with theimmobilized protein. Between different injections, the chip isregenerated using suitable regeneration buffer (such as glycine, pH2.5). The dissociation phase is fitted using Equation 1 to determineK_(off) (dissociate rate):R=R _(o)*exp(−K _(off) *t)  (1)The association phase is fitted using this value of K_(off) and Equation2 to determine K_(on) (association rate) and K_(D) (equilibriumconstant).

$\begin{matrix}{R = {\frac{R_{m\;{ax}}*C}{K_{D} + C}\left( {1 - {\exp\left( {{- \left( {{K_{on}*C} + K_{off}} \right)}t} \right)}} \right.}} & (2)\end{matrix}$where C represents either the antigen or antibody concentration insolution, R_(max) represents the saturation signal and t represents thetime.Epitope Binning: Surface Plasmon Resonance Assay

Epitope binning is performed using surface plasmon resonance assay, asdescribed above. One of the antibodies is immobilized on the surface ofthe chip. Recombinantly expressed human EGFR extracellular domain(EGFR-ECD) is then injected. As EGFR-ECD associates with the antibodyconjugated to the surface of the chip, the resonance signal increases.Sequential injections of antibodies that belong to the three bins 1, 2and 3 are performed. If the antibody binds overlapping epitopes with theinjected antibody, then the signal will not change compared to theprevious injection. If the antibody binds to a non-overlapping epitope,the signal on the chip will be higher than the previous injection. Theantibody conjugated to the chip is finally injected as free ligand toconfirm lack of binding with overlapping epitopes.

Cell Binding Assay

Cell binding assays for determining the K_(D) values are performed asfollows: A431 cells are detached with 3 mLs trypsin-EDTA at 37° C. for 5minutes. Complete DMEM (10 mLs) is added immediately to the trypsinizedcells, resuspended gently and spun down in a Beckman tabletop centrifugeat 1100 rpm for 5 minutes. Cells are resuspended in stain buffer(PBS+0.2% BSA+0.1% sodium azide) at a concentration of 2×10⁶ cells perml and 50 μl (1×10⁵ cells) aliquots are plated in a 96-well titer plate.

A 300 μl stock solution of 2000 nM anti-EGFR antibody is prepared instain buffer and 100 μl of it is serially diluted into 200 μl of stainbuffer. The concentrations of the diluted antibody range from 2000 nM to0.1 nM. 150 μl aliquots of the different protein dilutions are thenadded directly to the 50 μl cell suspension giving final concentrationsof 1500 nM, 500 nM, 166.7 nM, 55.6 nM, 18.5 nM, 6.17 nM, 2.05 nM, 0.68nM, 0.23 nM and 0.076 nM antibody.

Aliquoted cells in the 96-well plate are incubated with the antibodydilutions for 2 hr at room temperature with shaking and washed 3 timeswith 300 μl stain buffer. Cells are then incubated with 100 μl of a1:750 dilution of Alexa 647-labeled goat anti-human IgG in BD stainbuffer for 45 minutes with shaking at 4° C. Finally, cells are washedtwice, pelleted and resuspended in 250 μl stain buffer+0.5 μg/mlpropidium iodide. Analysis of 10,000 cells is done in a FACSCALIBUR flowcytometer using the FL4 channel. MFI values and the correspondingconcentrations of the anti-EGFR-antibodies are plotted on the y-axis andx-axis, respectively. The K_(D) of the molecule is determined usingGRAPHPAD PRISM software using the one-site binding model for anon-linear regression curve.

The K_(D) value is calculated based on the formula Y=Bmax*X/K_(D)+X(Bmax=fluorescence at saturation. X=antibody concentration. Y=degree ofbinding).

Measurement of EGFR Levels Via Immunoblotting

To prepare cell lysates, H1975 cells are trypsinized, harvested,counted, and plated in 6 well dishes at 1×10⁶ cells per well andincubated overnight to allow attachment to the culture plate. Cells arepre-treated with 1 μM concentration of P1X+P2X+P3X (P1X+P2X+P3X or P1X,P2X, & P3X indicates a combination at a 2:2:1 molar ratio of P1X, P2X,and P3X) for 1, 2, 5, and 24 hours before stimulation with rhEGF(Peprotech, cat#100-15) for 10 minutes. Cells are lysed with 100 μl ofMammalian Protein Extraction Reagent (Pierce, cat#78505). Proteinextraction reagent is supplemented with PhosSTOP (Roche,cat#04906837001) and Protease Inhibitor Cocktail tablets (Roche,cat#04693124001). Extracts of H1975 cells are denatured by boiling for 5minutes in sample buffer, subjected to reducing conditions, andelectrophoresed using SDS-PAGE 4-12% polyacrylamide gels for 50 minutesat 200V. Following transfer of proteins to a nitrocellulose membrane,nonspecific sites are blocked by incubation with Odyssey blocking buffer(LI-COR, cat#927-400-00) for one hour at room temperature. Membranes areincubated as required with mouse monoclonal anti-EGFR (1F4—labelingtEGFR; Cell Signaling, cat#2239); rabbit monoclonal anti-phospho44/42MAPK (Erk1/2, Thr202/Tyr204, D13.14.4E—labeling pERK; Cell Signaling,cat#4370); rabbit monoclonal anti-phosphoAKT (Ser473, 193H12; CellSignaling, cat#4058); rabbit monoclonal phospho-c-Jun (Ser73,D47G9—labeling p-c-Jun; Cell Signaling, cat#3270) and rabbit anti-PCNA(FL-261) (Santa Cruz Biotechnology, cat# sc-7907). Following overnightincubation with the primary antibodies, immunoblots are incubated withthe appropriate IRDye-labeled secondary antibody (IR800CW goatanti-mouse (Odyssey, cat#926-3210) or IR800CW goat anti-rabbit (Odyssey,cat#926-3211)) for 10 minutes and vacuumed through the membrane usingSNAP i.d., Protein Detection System (Millipore). Bands are detectedusing LI-COR Odyssey Infrared Imaging System and analyzed using Odysseysoftware.

Inhibition of Tumor Cell Proliferation In Vitro

Inhibition of cellular proliferation of cells expressing EGFR isexamined in vitro as follows: HCC827 and H1975 cancer cells are seededin 96 well tissue culture plates at 5,000 cells per well and grown inRPMI-1640 medium supplemented with antibiotics, 2 mM L-glutamine and 10%fetal calf serum (FCS) for 24 hours at 37° C. and 5% carbon dioxide.Medium is then switched to RPMI-1640 (with antibiotics, 2 mML-glutamine, 1% FBS) supplemented with 50 ng/mL EGF or 200 ng/ml AREG(amphiregulin; R&D Systems) in the presence of varying concentrations ofP1X+P2X+P3X or cetuximab (Bristol-Myers Squibb). Cell viability ismeasured using the CellTiter-Glo® (CTG) Luminescent Cell Viability Assay(Promega Corporation, cat# G7572) according to manufacturer'sinstructions. The CTG assay measures the number of viable cells inculture based upon quantitation of ATP present, which is an indicator ofmetabolically active cells. Control treatments include cells treatedwith RPMI-1640 with antibiotics, 2 mM L-glutamine, 1% FCS in thepresence (referred to as “+EGF” or “+AREG”) or absence (referred to as“−EGF” or “−AREG”) of 50 ng/mL EGF or 200 ng/ml AREG.

Inhibition of Tumor Cell Proliferation In Vitro with Drug Combinations

Inhibition of cellular proliferation of cells expressing EGFR withcombinations of therapeutic agents is examined in vitro as follows:indicated cancer cells are seeded into 96-well low-binding round-bottomtissue culture plates (Corning #7007) at the indicated cell number perwell and grown in RPMI-1640 medium supplemented with antibiotics, 2 mML-glutamine, 5 or 10% fetal calf serum (FCS) and indicated ligand(s),each at 5 nM, for 24 hours at 37° C. and 5% carbon dioxide. Therapeuticagents, alone or in combination, are then added in varyingconcentrations as indicated in figures in the same supplemented medium,and plates are allowed to incubate for 3 or 5 days at 37° C. and 5%carbon dioxide. Cell viability is measured using the CellTiter-Glo®(CTG) Luminescent Cell Viability Assay (Promega Corporation, cat# G7572)according to manufacturer's instructions. Control treatments includecells treated with RPMI-1640 with antibiotics, 2 mM L-glutamine, 5 or10% FCS and indicated ligands, each at 5 nM.

Inhibition of cell proliferation with combinations of P1X+P2X+P3X withMM-121 is examined by the above protocol with the followingmodifications: cells are seeded at 5,000 cells/well and RPMI-1640 mediumis supplemented with antibiotics, 2 mM glutamine, 5% FCS, 5 nM EGF and 5nM heregulin (hereafter “HRG”).

Inhibition of cell proliferation with combinations of P1X+P2X+P3X withdocetaxel (hereafter “DTX”) is examined by the above protocol with thefollowing modifications: cells are seeded at 5,000 cells/well andRPMI-1640 medium is supplemented with antibiotics, 2 mM glutamine, 5%FCS and 5 nM EGF.

Inhibition of cell proliferation with combinations of P1X+P2X+P3X withSN-38 is examined by the above protocol with the followingmodifications: cells are seeded at 2,500 cells/well and RPMI-1640 mediumis supplemented with antibiotics, 2 mM glutamine, 10% FCS and 5 nM EGF.

DU145 & H1975 Mouse Xenograft Studies

Nu/nu mice (Charles River Labs) are injected subcutaneously with cells.The resultant tumors are allowed to grow until they reach an averagesize of 300 mm³. Dosing is then initiated with the indicatedconcentrations of combinations of P1X with P2X, and P3X, cetuximab ormatched volume of PBS as vehicle control. Measurements are taken at 4day intervals and tumor volumes calculated using the formulaVolume=π/6×(W×L²). Cetuximab and P1X+P2X+P3X doses are normalized toprovide uniform serum exposures.

Efficacy of a combination of P1X with P2X, and P3X in vivo is assessedin a DU145 prostate cancer cell xenograft murine mouse model. 8×10⁶DU145 cells are injected subcutaneously into the flank of nu/nu mice.Once tumors reach an average size of 300 mm³, treatment is initiated.Groups of 10 mice are treated with either vehicle control (PBS); 2.075mg/kg of cetuximab, P1X with P2X, and P3X at component concentrationsthat were at a “murine ratio” designed to provide a 2:2:1 serum drugexposure for P1X, for P2X and for P3X, respectively, in the mouse, asfollows: P1X=2.53 mg/kg, P2X=7.26 mg/kg and P3X=0.66 mg/kg. Mice in thecetuximab group are dosed every four days. Mice in the P1X+P2X+P3X groupare dosed every two days. These doses and dosing intervals are chosen togive an equivalent serum exposure of cetuximab and the murine ratioantibody trio.

Patient Derived Colorectal Cancer Tumor Xenograft Study

The following example employs a bin 1 antibody, “ca”, which is theparental antibody to the bin 1 antibody P1X. This antibody is describedin co-pending patent application “Antibodies Against Epidermal GrowthFactor Receptor (EGFR) and Uses Thereof”, Publication No. US2011-0287002, which is herein incorporated by reference.

Efficacy of a combination of ca+P2X+P3X in vivo is assessed in a patientderived colorectal cancer tumor xenograft murine mouse model (PDX model#ST094, South Texas Accelerated Research Therapeutics Discovery). Inthese human xenograft in mouse host experiments, antibody ca wassubstituted for antibody P1X because P1X, but not ca, cross reacts with(binds to) mouse EGFR as well as binding to human EGFR, and the bindingof P1X to the mouse antigen depletes the amount of this antibody in themouse circulation, altering exposure of the human tumor to the antibody.Results obtained with ca are predictive of results that would beobtained in human patients with P1X, except that the antibody triocomprising P1X would be expected to be more active than the triocomprising ca in its stead.

Fragments of tumors are implanted subcutaneously into the flank offemale immune-deficient mice. Once tumors reach an average size of125-225 mm³, treatment is initiated. Groups of 10 mice are treated byintraperitoneal injection with either vehicle control (PBS); 100 mg/kgirinotecan; ca+P2X+P3X at component concentrations that were at a“murine ratio” designed to provide a 2:2:1 serum drug exposure for ca,for P2X and for P3X, respectively, in the mouse, as follows: for thefirst (“loading”) dose, ca=40 mg/kg, P2X=28 mg/kg and P3X=20 mg/kg; forthe remaining (“maintenance”) doses, ca=20 mg/kg, P2X=28 mg/kg andP3X=10 mg/kg. Mice in the irinotecan group are dosed on Days 0, 7, and14. Mice in the ca+P2X+P3X group are dosed every seven days with ca+P3Xand three times per week with P2X. Mice in the [ca+P2X+P3X]+irinotecancombination group are dosed in the same manner as listed for therespective monotherapy groups. Measurements are taken twice weekly andtumor volumes calculated using the formula Volume=π/6×(W²×L).

Ligand Antagonism Cell Binding Assay

Cell binding assays for determining the binding of EGF ligand in thepresence of single or multiple antibodies are performed as follows: A431cells are detached with 5 mL trypsin-EDTA at 37° C. for 5 minutes.Complete DMEM (10 mL) is added immediately to the trypsinized cells,resuspended gently and spun down in a Beckman tabletop centrifuge at1200 rpm for 7 minutes. Cells are resuspended in stain buffer (PBS+2%FBS+0.1% sodium azide) at a concentration of 3×10⁵ cells per ml and 100μl (3×10⁴ cells) aliquots are plated in a 96-well titer plate.

A 5 mL 2× stock solution of anti-EGFR antibody is prepared in stainbuffer at the concentrations indicated in each example. A 10 mL 3× stocksolution of recombinant human EGF ligand conjugated to a biotin tag(biotin-EGF) is prepared in stain buffer and 100 μl of it is seriallydiluted into 200 μl of stain buffer. The concentrations of the dilutedbiotin-EGF range from 600 nM to 9 pM. 100 μl aliquots of the anti-EGFRantibody are then added directly to the 100 μl cell suspension giving afinal concentration as indicated in each example. Aliquoted cells in the96-well plate are incubated with the antibody dilutions for 1 hr at roomtemperature. 100 μl aliquots of the biotin-EGF are then added directlyto the 100 μl cell suspension giving a final concentration of 200 nM,66.67 nM, 22.22 nM, 7.41 nM, 2.47 nM, 0.82 nM, 0.27 nM, 0.09 nM, 0.03nM, 0.01 nM, and 0.003 nM biotin-EGF. Aliquoted cells in the 96-wellplate are incubated with the antibody and biotin-EGF dilutions for 10min at room temperature, washed 1 time with 100 μl stain buffer, andthen spun down in a Beckman tabletop centrifuge at 1200 rpm for 7minutes. Cells are resuspended in 100 μl of a 1:500 dilution of AlexaFluor® 647 Streptavidin conjugate (Invitrogen Life Technologies) instain buffer for 30 minutes at room temperature in the dark. Finally,cells are washed twice with 100 μl stain buffer, pelleted andresuspended in 80 μl fixing buffer (PBS+2% FBS+2% paraformaldehyde) andtransferred to 96-well U-bottom Assay Plates (Becton Dickinson) andsealed with foil and stored at 4° C.

Analysis of 10,000 cells is done in a FACSCALIBUR flow cytometer usingthe FL4 channel. Data are analyzed using WinList 6.0 software. MeanFluorescence Intensity (MFI) values and the corresponding concentrationsof the biotin-EGF ligand are plotted on the y-axis and x-axis,respectively.

Example 1 Epitope Mapping/Binning

Antibodies P1X, P2X, and P3X were generated via affinity maturation ofparental antibodies. Respective parental antibodies ca, cd, and ch aredisclosed in copending patent application Serial No. PCT/US2011/3528.Epitope mapping and binning experiments were performed to demonstratethat P1X, P2X, and P3X share the same non-overlapping epitopes as theirrespective parental molecules.

The Bins were designed so that selected antibodies would span threedistinct, non-overlapping epitopes on the extracellular domain (ECD) ofEGFR. These are grouped into three bins: Bin1 is mapped to Domain III ofEGFR and represents the c225 epitope (the site of cetuximab binding);Bin2 is mapped to Domain I and represents the ICR10 epitope (AbcamAb231) (Cochran et al. (2004) Journal of Immunological Methods,287:147-158); and Bin3 is mapped to Domain III and represents the cloneH11 epitope (Spangler J. et al. PNAS. 107: 13252-13257, 2010). Bin1(B1-7MT-Ala) and Bin3 (B3-4MT) mutants were generated for epitopemapping at the amino acid positions shown below in bold in the EGFRextracellular domain.

EGFR ECD (SEQ ID NO: 33) 1MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV 61VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA 121VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF 181QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC 241TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV 301VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK 361NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF 421ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL 481FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCN 541LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM 601GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSHHHHH HThe following substitution mutants were made at the bolded amino acidpositions using standard recombinant DNA technology to create the Bin1(B1) and Bin3 (B3) epitope mutants with the following mutant residues:

Mutants

Bin1 (B1) Mutant Residues

B1-7MT-Ala: Q408A, Q432M, H433E, K467A, K489A, I491A, N497A

Bin3 (B3) Mutant Residues

B3-4MT: S380A, F381G, T382A, H383G

A direct ELISA was performed using EGFR-ECD wild-type (WT), a Bin1epitope mutant (c225 epitope), or a Bin3 epitope mutant (H11 epitope) ascapture reagents. Varying concentrations (1, 0.25, 0.06, and 0.02 μg/ml)of monoclonal antibodies (mAbs) P1X (Bin1), P2X (Bin2), and P3X (Bin3)were incubated with the capture reagents at room temperature for 2hours, followed by detection with HRP-conjugated anti-human Fcpolyclonal antibody for 1 hour. As shown in FIG. 1A, all threeantibodies bound to the WT extracellular domain of EGFR, whereas theBin1 antibody P1X did not bind to the Bin1 mutant epitope, and the P3Xantibody did not bind to the Bin3 mutant epitope.

A surface plasmon resonance experiment was performed to demonstrate thatP2X associates to the ICR10 epitope on Domain I of EGFR extraceullulardomain (FIG. 1B). ICR10 was conjugated to the surface of a BIACORE chip.0.5 μM EGFR-ECD was injected followed by sequential injections of 0.5 μMof antibodies P1X (Bin1), P2X (Bin2), and P3X (Bin3). While P1X and P3Xare observed to simultaneously associate with ICR10-bound EGFR-ECD, P2Xis shown to not associate.

In order to demonstrate that the epitopes for P1X, P2X, and P3X aredistinct and non-overlapping, a series of three surface plasmonresonance binning experiments were performed. P1X (FIG. 2A), P2X (FIG.2B), or P3X (FIG. 2C) were conjugated to the surface of a BIACORE chip.0.5 μM EGFR-ECD was injected followed by sequential injections of 0.5 μMof antibodies P3X, P2X, and P1X. Injection of the same antibody asconjugated on the BIACORE chip serves as a negative control. In allthree experiments, the two antibodies from the remaining bins areobserved to associate with EGFR-ECD. Thus, the results of the threeexperiments demonstrate that P1X, P2X, and P3X have non-overlapping,distinct epitopes and can simultaneously associate with EGFR-ECD.

Example 2 Binding Affinities

The monovalent affinities of P1X, P2X, and P3X to EGFR were measured byKinExA. Data are shown below in Table 1. Affinities of P1X, P2X, and P3Xare all better than 0.4 nM and are all improved relative to the parentmolecules. The affinity of P1X (11 pM) is 13.18 times better than theBin 1 parent molecule ca (145 pM). The affinity of P2X (70 pM) is 7.71times better than the Bin 2 parent molecule cd (540 pM). The affinity ofP3X (360 pM) is 2.10 times better than the Bin 3 parent molecule ch (757pM).

TABLE 1 Association Rate Dissociation Rate Antibody (1/M * sec) (1/sec)K_(D) (M) P1X 3.73E+06 4.10E−05 1.10E−11 ca (P1X parent) N.D. N.D.1.45E−10 P2X 7.06E+05 4.94E−05 7.00E−11 cd (P2X parent) 9.62E+055.20E−04 5.40E−10 P3X 1.16E+06 4.16E−04 3.60E−10 ch (P3X parent)5.87E+05 4.44E−04 7.57E−10

Example 3 Cell Binding Assays with Single Antibodies

A cell binding assay was performed to demonstrate that monoclonalantibodies P1X, P2X, and P3X can associate with EGFR on A431 cells (FIG.3). A431 cells were incubated with a dilution series of single antibodyfor 2 hr and the amount of bound antibody measured by quantitative flowcytometry, as described in the methodology section above. Theconcentrations used in the dilution series for the antibodies are shownbelow in Table 2. The ordinarily skilled artisan will understand thateach specific concentration value in Table 2 is subject to some minorexperimental variability, so that each specific concentration givenindicates a value of about the indicated concentration (e.g., aconcentration indicated in a table as 0.1 nM represents a value of about0.1 nM).

TABLE 2 Conc, Log(Molar) Conc, nM −7.00 100.00 −7.48 33.33 −7.95 11.11−8.43 3.70 −8.91 1.23 −9.39 0.41 −9.86 0.14 −10.34 0.05 −10.82 0.02−11.29 0.01 −11.77 0.00

The on-cell binding affinities under these experimental conditions werecalculated, via regression to a 4 parameter logistic equation usingGraphPad Prism® software, to be 168 pM (P1X), 340 pM (P2X) and 748 pM(P3X).

Example 4 Phospho-EGF Receptor Signaling Inhibition by Single Antibodies

A431 cells were treated with single antibodies and their ability toinhibit EGF-dependent phospho-EGFR activity was measured by phospho-EGFRELISA. P1X and P2X potently inhibit phospho-EGFR activity in adose-dependent manner, with respective IC50 values of 3.09 nM and 4.19nM, while treatment with P3X elicits partial phospho-EGFR inhibition(FIG. 4). The concentrations used in the dilution series for theantibodies are shown below in Table 3. The ordinarily skilled artisanwill understand that each specific concentration value in Table 3 issubject to some minor experimental variability, so that each specificconcentration given indicates a value of about the indicatedconcentration (e.g., a concentration indicated in a table as 0.1 nMrepresents a value of about 0.1 nM).

TABLE 3 Conc, Log(Molar) Conc, nM −6.60 250.00 −7.20 62.50 −7.81 15.62−8.41 3.91 −9.01 0.98 −9.61 0.24 −10.21 0.06 −10.82 0.02 −11.42 0.00

Example 5 Phospho-ERK Signaling Inhibition by Single and PairwiseCombinations of Antibodies and Comparison to Parental Antibodies

A431 cells were treated with a dilution series of single P1X or caantibody and phospho-ERK inhibition measured by phospho-ERK ELISA (FIG.5A). P1X elicited dose-dependent inhibition of phospho-ERK activitywhile the ca parental antibody elicited only partial inhibition.

A431 cells were treated with a dilution series of pairwise combinationsof P1X+P3X or their respective parent antibodies ca+ch and phospho-ERKinhibition measured by phospho-ERK ELISA (FIG. 5B). Both combinationsinhibit phospho-ERK generation in a dose-dependent manner, but thecombination of P1X+P3X provides superior inhibition. The combination ofP1X+P3X elicited 82% inhibition of phospho-ERK activity while theparental combination elicited only 71%, as calculated by a fit to a 4parameter logistic equation using GraphPad Prism software.

A431 cells were treated with a dilution series of pairwise combinationsof P1X+P2X or their respective parent antibodies ca+cd and phospho-ERKinhibition measured by phospho-ERK ELISA (FIG. 5C). Both combinationsinhibit phospho-ERK generation in a dose-dependent manner, but thecombination of P1X+P2X demonstrates an observable improvement in theIC90 value versus the parental combination.

The concentrations used in the dilution series for the antibodies areshown below in Table 4 (for the data in FIG. 5A) and in Table 5 (for thedata in FIGS. 5B and 5C). The ordinarily skilled artisan will understandthat each specific concentration value in Tables 4 and 5 is subject tosome minor experimental variability, so that each specific concentrationgiven indicates a value of about the indicated concentration (e.g., aconcentration indicated in a table as 0.1 nM represents a value of about0.1 nM).

TABLE 4 Conc, Log(Molar) Conc, nM −5.70 2000.00 −6.18 666.67 −6.65222.22 −7.13 74.07 −7.61 24.69 −8.21 6.17 −8.81 1.54 −9.41 0.39 −10.020.10

TABLE 5 Conc, Log (Molar) Conc, nM −5.69897 2000.00 −6.176091 666.67−6.653213 222.22 −7.130334 74.07 −7.607455 24.69 −8.209515 6.17−8.811575 1.54 −9.413635 0.39 −10.01569 0.10 −10.61775 0.02

It is noted that the concentrations shown in Table 5 are totalconcentrations for the pairs of antibodies used. The ratio used is 1:1so each individual antibody in the pair comprises half of the totalconcentration.

Example 6 Phospho-ERK Signaling Inhibition by Different CombinationRatios of P1X, P2X, and P3X

A431 cells were treated with a dilution series of P1X and phospho-ERKinhibition measured by ELISA (FIG. 6A). The concentrations used in thedilution series for the antibodies are shown below in Table 6. Theordinarily skilled artisan will understand that each specificconcentration value in Table 6 is subject to some minor experimentalvariability, so that each specific concentration given indicates a valueof about the indicated concentration (e.g., a concentration indicated ina table as 0.1 nM represents a value of about 0.1 nM).

TABLE 6 Conc, Log (Molar) Conc, nM −5.69897 2000.00 −6.176091 666.67−6.653213 222.22 −7.130334 74.07 −7.607455 24.69 −8.084577 8.23−8.561698 2.74 −9.038818 0.91 −9.51594 0.30 −9.993061 0.10

The experiment was carried out as described in the methodology section.Under these experimental conditions, P1X inhibits 81% of phospho-ERKactivity at saturating doses with an IC50 value of about 25 nM (27 nM).Therefore the approximate location on the plot of the IC50 is indicatedin FIG. 6A by “25 nM” and 25 nM was set as the constant concentration ofP1X in the following experiments.

A431 cells were treated with dilution series of 5 combination ratios ofP3X+P2X in combination with a constant P1X concentration of 25 nM andphospho-ERK inhibition measured by ELISA (FIG. 6B). The concentrationsused in the dilution series for the antibodies are shown below in Table7. The ordinarily skilled artisan will understand that each specificconcentration value in Table 7 is subject to some minor experimentalvariability, so that each specific concentration given indicates a valueof about the indicated concentration (e.g., a concentration indicated ina table as 0.1 nM represents a value of about 0.1 nM).

TABLE 7 Conc, Log (Molar) Conc, nM −5.69897 2000.00 −6.176091 666.67−6.653213 222.22 −7.130334 74.07 −7.607455 24.69 −8.084577 8.23−8.561698 2.74 −9.038818 0.91 −9.51594 0.30 −9.993061 0.10It is noted that the concentrations shown in Table 7 are totalconcentrations for P2X+P3X. The individual concentration of P2X and P3Xis dependent on the indicated ratio. The experiment was carried out asdescribed in the methodology section. The ratios of P3X:P2X used were1:0, 0:1, 1:2, 2:1, and 1:1. All ratios inhibited greater than 70% ofphospho-ERK activity, with those combinations containing all threeantibodies providing the highest degree of inhibition.

A431 cells were treated with dilution series of 6 combination ratios ofP1X:P2X:P3X and phospho-ERK inhibition measured by ELISA (FIG. 6C). Theconcentrations used in the dilution series for the antibodies are shownbelow in Table 8. The ordinarily skilled artisan will understand thateach specific concentration value in Table 8 is subject to some minorexperimental variability, so that each specific concentration givenindicates a value of about the indicated concentration (e.g., aconcentration indicated in a table as 0.1 nM represents a value of about0.1 nM).

TABLE 8 Ratio (P1X:P2X:P3X) LogM nM 1:0.01:1 1:0.1:1 1:1:1 2:0.1:12:0.1:1 2:2:1 −10.8148 0.0153 X −10.8141 0.0153 X −10.7958 0.0160 X−10.7889 0.0163 X −10.6409 0.0229 X −10.5951 0.0254 X −10.3377 0.0460 X−10.3370 0.0460 X −10.3187 0.0480 X −10.3118 0.0488 X −10.1638 0.0686 X−10.1180 0.0762 X −9.8606 0.1379 X −9.8598 0.1381 X −9.8415 0.1440 X−9.8347 0.1463 X −9.6866 0.2058 X −9.6409 0.2286 X −9.3834 0.4136 X−9.3827 0.4143 X −9.3644 0.4321 X −9.3576 0.4390 X −9.2095 0.6173 X−9.1638 0.6859 X −8.9063 1.2407 X −8.9056 1.2428 X −8.8873 1.2963 X−8.8805 1.3169 X −8.7324 1.8519 X −8.6866 2.0576 X −8.4292 3.7222 X−8.4285 3.7284 X −8.4102 3.8889 X −8.4033 3.9506 X −8.2553 5.5555 X−8.2095 6.1728 X −7.9521 11.1667 X −7.9514 11.1852 X −7.9331 11.6667 X−7.9262 11.8518 X −7.7782 16.6667 X −7.7324 18.5185 X −7.4750 33.5000 X−7.4742 33.5555 X −7.4559 35.0000 X −7.4491 35.5556 X −7.3010 50.0000 X−7.2553 55.5556 X −6.9978 100.5000 X −6.9971 100.6667 X −6.9788 104.9999X −6.9720 106.6665 X −6.8239 149.9999 X −6.7782 166.6668 X −6.5207301.4998 X −6.5200 302.0000 X −6.5017 315.0003 X −6.4949 320.0000 X−6.3468 450.0005 X −6.3010 500.0000 XThe experiment was carried out as described in the methodology section.The ratios of P1X:P2X:P3X used were 1:0.01:1, 1:0.1:1, 1:1:1, 2:0.01:1,2:0.1:1, and 2:2:1. All ratios inhibited greater than 70% of phospho-ERKactivity.

A431 cells were treated with dilution series of 5 combination ratios ofP1X:P2X and phospho-ERK inhibition measured by ELISA (FIG. 6D). Theconcentrations used in the dilution series for the antibodies are shownbelow in Table 9. The ordinarily skilled artisan will understand thateach specific concentration value in Table 9 is subject to some minorexperimental variability, so that each specific concentration givenindicates a value of about the indicated concentration (e.g., aconcentration indicated in a table as 0.1 nM represents a value of about0.1 nM).

TABLE 9 Conc, Log (Molar) Conc, nM −6.522879 300.00 −7 100.00 −7.47712133.33 −7.954243 11.11 −8.431364 3.70 −8.908485 1.23 −9.385606 0.41−9.862727 0.14 −10.33985 0.05 −10.81697 0.02

It is noted that the concentrations shown in Table 9 are totalconcentrations for P1X+P2X. The individual concentration of P1X and P2Xis dependent on the indicated ratio. The experiment was carried out asdescribed in the methodology section. The ratios of P1X:P2X used were1:2, 5:1, 9:1, 50:1, and 1:5. All ratios except 1:5 (P1X:P2X) inhibitedgreater than 70% of phospho-ERK activity within the concentration rangeused in the experiment. However, fitting a 4 parameter logisticinhibition curve to the 1:5 ratio (P1X:P2X) data predicts that thiscombination will achieve 70% phospho-ERK inhibition at a concentrationof 35.7 nM, marginally higher than the dose used in the experiment andwell within the range achievable under physiological conditions.

Example 7 Phospho-EGFR and Phospho-ERK Signaling Inhibition by a 2:2:1Ratio Combination of P1X, P2X, and P3X

A431 cells were treated with a dilution series of a 2:2:1 molar ratiocombination of antibodies P1X, P2X, and P3X (this combination at thismolar ratio is referred to herein as “P1X+P2X+P3X”) and phospho-EGFR andphospho-ERK inhibition measured by ELISA (FIG. 7A). The concentrationsused in the dilution series for the antibodies are shown below in Table10. The ordinarily skilled artisan will understand that each specificconcentration value in Table 10 is subject to some minor experimentalvariability, so that each specific concentration given indicates a valueof about the indicated concentration (e.g., a concentration indicated ina table as 0.1 nM represents a value of about 0.1 nM).

TABLE 10 Conc, Log (Molar) Conc, nM −6.30 500.00 −6.78 166.67 −7.2655.56 −7.73 18.52 −8.21 6.17 −8.69 2.06 −9.16 0.69 −9.64 0.23 −10.120.08 −10.60 0.03

It is noted that the concentrations shown in Table 10 are totalconcentrations for P1X+P2X+P3X. The ratio used is 2:2:1, so theindividual concentrations of P1X, P2X and P3X are 40%, 40% and 20%,respectively. Experiments were carried out as described in themethodology section. The combination of three antibodies is a potentinhibitor of both phospho-EGFR and phospho-ERK activities, withrespective IC50 values of 2.30 nM and 9.87 nM.

P1X+P2X+P3X was compared to P1X single (FIG. 7B) and P2X single (FIG.7C). A431 cells were treated with a dilution series of antibody andphospho-ERK inhibition measured by ELISA. The concentrations used in thedilution series for the antibodies for the experiments shown in FIGS. 7Band 7C are shown below in Table 11. The ordinarily skilled artisan willunderstand that each specific concentration value in Table 11 is subjectto some minor experimental variability, so that each specificconcentration given indicates a value of about the indicatedconcentration (e.g., a concentration indicated in a table as 0.1 nMrepresents a value of about 0.1 nM).

TABLE 11 Conc, Log (Molar) Conc, nM −5.69897 2000.00 −6.176091 666.67−6.653213 222.22 −7.130334 74.07 −7.607455 24.69 −8.084577 8.23−8.561698 2.74 −9.038818 0.91 −9.51594 0.30 −9.993061 0.10

It is noted that the concentrations shown in Table 11 are totalconcentrations for P1X+P2X+P3X. The ratio used is 2:2:1, so theindividual concentrations of P1X, P2X and P3X are 40%, 40% and 20%,respectively. Experiments were carried out as described in themethodology section with the exception that 80 nM of EGF ligand was usedto stimulate cells. The 2:2:1 ratio combination of antibodies is apotent inhibitor of phospho-ERK activity compared to P1X and P2X, whichrespectively provide partial and no inhibition.

Example 8 EGF Receptor Down-Regulation and Inhibition of pERK, pAKT andp-c-Jun Signaling in H1975 Cells Following Treatment with P1X+P2X+P3X

Cells were pre-incubated for 2 hours with of P1X+P2X+P3X equaling 1 μMtotal antibody prior to stimulation with 50 ng/ml rhEGF (PeproTech) for10 minutes, as described in the methodology section above. Immunoblotsof cell lysates were separately probed with antibodies against tEGFR,pERK, pAKT or p-c-Jun and densitometry of the bands was normalized tothe loading control PCNA and to lysates of control untreated cells. EGFreceptor down-regulation in response to P1X+P2X+P3X treatment is shownin FIG. 8A and inhibition of pERK, pAKT and p-c-Jun signaling inresponse to P1X+P2X+P3X treatment is shown in FIG. 8B.

Example 9 Inhibition of Tumor Cell Proliferation In Vitro

Inhibition of tumor cell proliferation in vitro was analyzed by themethods described above or minor variations thereof. The non-small celllung cancer (NSCLC) lines HCC827 and H1975 were plated at 5000cells/well and treated with antibody combinations ranging from 0.1-1 μM(final concentration). FIGS. 9A-9D show inhibition of cell proliferationusing CellTiter-Glo® (CTG) Luminescent Cell Viability Assay (PromegaCorporation) that measures the number of viable cells in culture basedupon quantitation of ATP present, which is an indicator of metabolicallyactive cells. FIGS. 9A and 9B show potent inhibition of growth of HCC827and H1975 cells over a range of P1X+P2X+P3X concentrations, but not bycetuximab treatment or assay medium alone (1% FCS) in the presence ofEGF ligand. FIGS. 9C and 9D show potent inhibition of growth of HCC827and H1975 cells over a range of concentrations for both P1X+P2X+P3X andcetuximab, but not by assay medium alone (1% FCS) in the presence ofAREG ligand. These results demonstrate the ability of P1X+P2X+P3X toinhibit tumor cell proliferation in vitro in response to bothhigh-affinity (EGF) and low-affinity (AREG) ligands, whereas cetuximabis only effective in cells treated with low-affinity (AREG) ligand. Theconcentrations used in the dilution series for the antibodies for theexperiments shown in FIGS. 9A-D are shown below in Table 12. Theordinarily skilled artisan will understand that each specificconcentration value in Table 12 is subject to some minor experimentalvariability, so that each specific concentration given indicates a valueof about the indicated concentration (e.g., a concentration indicated ina table as 0.1 nM represents a value of about 0.1 nM).

TABLE 12 Conc, Log (Molar) Conc, nM −6.000000 1000.00 −6.301030 500.00−6.602060 250.00 −6.903090 125.00 −7.204120 62.50 −7.806180 15.63−8.408240 3.91 −9.010300 0.98 −9.913390 0.12

It is noted that the concentrations shown in Table 12 are totalconcentrations for P1X+P2X+P3X. The ratio used is 2:2:1, so theindividual concentrations of P1X, P2X and P3X are 40%, 40% and 20%,respectively.

Example 10 Inhibition of Tumor Growth In Vivo

Efficacy of P1X+P2X+P3X in vivo was assessed in a H1975 lung cancer cellxenograft murine mouse model. 2×10⁶ NCI-H1975 cells were injectedsubcutaneously into the flank of nu/nu mice. Once tumors had reached anaverage size of 300 mm³ treatment was initiated. Groups of 10 mice weretreated with either vehicle control (PBS); or the murine ratio antibodytrio at the following component concentrations: murine ratio antibodytrio-Low P1X=2.53 mg/kg, P2X=7.26 mg/kg and P3X=0.66 mg/kg or murineratio antibody trio-Medium P1X=5.06 mg/kg, P2X=14.52 mg/kg and P3X=1.33mg/kg. Mice were treated every two days.

The results shown in FIG. 10A (DU145 xenograft model) and FIG. 10B(H1975 xenograft model) demonstrate the ability of P1X+P2X+P3X toinhibit tumor growth (and, inferentially, cell proliferation) in vivo.

Example 11 Ligand Antagonism Cell Binding Assays with Single Antibodies

A cell binding assay was performed to demonstrate that monoclonalantibodies P1X, P2X, and P3X can antagonize the interaction of EGFligand and EGF receptor on A431 cells. A431 cells were incubated withone dose of single antibody for 1 hr followed by a dilution series ofbiotin-EGF ligand and the amount of bound biotin-EGF ligand measured byquantitative flow cytometry, as described in the methods section above.The concentrations for the antibodies are shown below in Table 13 andrepresent a sub-saturating concentration (approx. EC90 concentration) ofcell binding as determined from the analysis demonstrated in Example 3.The concentrations used in the dilution series for the biotin-EGF areshown below in Table 14. The values in Table 13 and Table 14 are subjectto some minor experimental variability, so that each specificconcentration given indicates a value of about the indicatedconcentration (e.g., a concentration indicated in a table as 0.1 nMrepresents a value of about 0.1 nM).

TABLE 13 Antibody Conc, Log (Molar) Conc, nM P1X −9.01 0.97 P2X −8.702.00 P3X −8.33 4.68

TABLE 14 Conc, Log (Molar) Conc, nM −6.70 200 −7.18 66.67 −7.65 22.22−8.13 7.41 −8.61 2.47 −9.08 0.82 −9.56 0.27 −10.04 0.09 −10.52 0.03−11.00 0.01 −11.52 0.003

The results are shown in FIG. 11, which demonstrates that each of thesingle antibodies (P1X, P2X and P3X) alone are capable of antagonizingthe interaction of EGF ligand and EGF receptor on A431 cells, with P1Xand P2X exhibiting more potent inhibitory activity than P3X.

Example 12 Ligand Antagonism Cell Binding Assays with Single andCombinations of Antibodies or Fabs

A cell binding assay was performed to determine the extent to whichsingle antibodies and multiple antibody combinations of monoclonalantibodies P1X, P2X, and P3X and single and multiple combinations ofmonovalent Fab fragments P1X Fab, P2X Fab, and P3X Fab can antagonizethe interaction of EGF ligand and EGF receptor on A431 cells. A431 cellswere incubated with one dose of antibody or Fab for 1 hr followed by adilution series of biotin-EGF ligand and the amount of bound biotin-EGFligand measured by quantitative flow cytometry, as described in themethodology section above. The concentration of antibodies and Fab was10 nM. The combinations of three antibodies (P1X+P2X+P3X) and threeantibodies (P1X Fab+P2X Fab+P3X Fab) were formulated in a ratio of 2:2:1and were dosed at a total concentration of 10 nM. The concentrationsused in the dilution series for the biotin-EGF are shown above in Table14. The ordinarily skilled artisan will understand that each specificconcentration value in Table 14 is subject to some minor experimentalvariability, so that each specific concentration given indicates a valueof about the indicated concentration (e.g., a concentration indicated ina table as 0.1 nM represents a value of about 0.1 nM).

The results are shown in FIG. 12A (single and combinations of monoclonalantibodies P1X, P2X, and P3X) and FIG. 12B (single and combinations ofmonovalent Fab fragments P1X Fab, P2X Fab, and P3X Fab). The results inFIG. 12A demonstrate that again all three antibodies alone were capableof antagonizing the interaction of EGF ligand and EGF receptor on A431cells, with P1X and P2X exhibiting more potent inhibitory activity thanP3X, and the triple combination of P1X+P2X+P3X also showed potentinhibitory activity. The results in FIG. 12B demonstrate that P1X Fabshowed the strongest inhibitory activity alone, with P3X Fab aloneshowing intermediate inhibitory activity alone and P2X Fab showing onlyminimal inhibitory activity alone. The triple combination of P1X Fab+P2XFab+P3X Fab also showed strong inhibitory activity, although less potentthan P1X Fab alone.

Example 13 Phospho-EGFR and Phospho-ERK Signaling Inhibition by a 2:2:1Molar Ratio Combination of P1X, P2X, and P3X Antibodies or Fabs

A431 cells were treated with a dilution series of a 2:2:1 molar ratiocombination of antibodies P1X, P2X, and P3X (“P1X+P2X+P3X”) or Fabs P1XFab, P2X Fab, P3X Fab (this combination at this molar ratio is referredto herein as “P1X Fab+P2X Fab+P3X Fab”) and phospho-EGFR and phospho-ERKinhibition measured by ELISA. Experiments were performed with rhEGF(PeproTech) dosed at a concentration of 50 ng/mL or 500 ng/mL. Theconcentrations used in the dilution series for the antibodies and Fabsare shown below in Table 15. The ordinarily skilled artisan willunderstand that each specific concentration value in Table 15 is subjectto some minor experimental variability, so that each specificconcentration given indicates a value of about the indicatedconcentration (e.g., a concentration indicated in a table as 0.1 nMrepresents a value of about 0.1 nM).

TABLE 15 Conc, Log (Molar) Conc, nM −5.70 2000.00 −−6.18 666.67 −6.65222.22 −7.13 74.07 −7.61 24.70 −8.08 8.23 −8.56 2.74 −9.04 0.91 −9.520.30 −9.99 0.10

The results are shown in FIGS. 13A-D, wherein FIGS. 13A and 13B show theresults of the phospho-EGFR inhibition assay and FIGS. 13C and 13D showthe results of the phosphor-ERK inhibition assay, with FIGS. 13A and 13Cshowing the results at low doses (50 ng/ml or 8 nM) and with FIGS. 13Band 13D showing the results at high doses (500 ng/ml or 80 nM).

With respect to inhibition of phospho-EGFR, the results in FIGS. 13A and13B demonstrate that both triple combinations, P1X+P2X+P3X mAbs and P1XFab+P2X Fab+P3X Fab fragments, exhibited strong inhibition at both thelow dose and the high dose tested.

With respect to inhibition of phospho-ERK, the results in FIGS. 13C and13D demonstrate that both triple combinations, P1X+P2X+P3X mAbs and P1XFab+P2X Fab+P3X Fab fragments, exhibited inhibition at both the low doseand the high dose tested, with the P1X+P2X+P3X mAb combinationexhibiting more potent inhibition that the P1X Fab+P2X Fab+P3X Fabfragment combination.

Example 14 EGF Receptor Down-Regulation in DU-145 Cells FollowingTreatment with P1X+P2X+P3X, P1X Fab+P2X Fab+P3X Fab, or Cetuximab

Cells were pre-incubated for 2, 6, or 24 hours with of P1X+P2X+P3X, P1XFab+P2X Fab+P3X Fab, or cetuximab equaling 50 nM, 100 nM, and 50 nM,respectively. The Fab combination is dosed at twice the concentration asP1X+P2X+P3X and cetuximab to account for a single binding moiety on aFab molecule versus two binding moieties on and IgG molecule.Immunoblots of cell lysates were probed with antibodies against totalEGFR (tEGFR) and the pcna housekeeping protein as a control, asdescribed in the methodology section above. EGF receptor down-regulationin response to treatment is shown in FIG. 14. The results demonstratethat treatment with P1X+P2X+P3X led to observable down-regulation ofEGFR, in a time dependent manner, whereas treatment with P1X Fab+P2XFab+P3X Fab or cetuximab did not lead to observable down-regulation ofEGFR on visual inspection of the immunoblots.

Example 15 Inhibition of Tumor Growth and Cell Proliferation In Vivo inCombination with Irinotecan

Efficacy of [ca+P2X+P3X], irinotecan and [ca+P2X+P3X]+irinotecan wereassessed in vivo compared to vehicle control (PBS) in a patient-derivedcolorectal cancer tumor xenograft murine mouse model by the methodsdescribed above. Arrows indicate days on which irinotecan wasadministered. The results shown in FIG. 15 demonstrate the ability of[ca+P2X+P3X] and of the combination of [ca+P2X+P3X] and irinotecan toinhibit tumor cell proliferation in vivo, with respect to vehiclecontrol. The results shown in FIG. 15 also demonstrate the ability ofthe combination of [ca+P2X+P3X] and irinotecan to inhibit tumor growth(and cell proliferation) in vivo to a greater extent than either[ca+P2X+P3X] or irinotecan alone.

Example 16 Inhibition of Tumor Cell Proliferation In Vitro byCombinations of [P1X+P2X+P3X] and MM-121

Inhibition of tumor cell proliferation in vitro was analyzed in the celllines A549, BxPC-3, DU 145, NCI-H1355, NCI-H226, NCI-H322M, NCI-H358,NCI-H520, HCC827, HT-1197, RT-112, SCaBER, SK-MES-1 and SW 900 by themethods described above. Cells were treated with all 80 pairwisecombinations of [P1X+P2X+P3X] and MM-121 antibody concentrations listedin Table 16.

TABLE 16 P1X + P2X + P3X Conc, nM MM-121 Conc, nM 2000 2000 666.7 666.7222.2 222.2 74.1 74.1 24.7 24.7 8.2 8.2 2.7 2.7 0.9 0 0.3 0

It is noted that the concentrations shown in Table 16 are totalconcentrations for P1X+P2X+P3X. The ratio used is 2:2:1, so theindividual concentrations of P1X, P2X and P3X are 40%, 40% and 20% ofthe total concentration, respectively. The ordinarily skilled artisanwill understand that each specific concentration value in Table 16 issubject to some minor experimental variability, so that each specificconcentration given indicates a value of about the indicatedconcentration (e.g., a concentration indicated in a table as 2000 nMrepresents a value of about 2000 nM).

For each cell line, inhibition of cell proliferation was measured usingthe CellTiter-Glo® (CTG) Luminescent Cell Viability Assay (PromegaCorporation) which measures the number of viable cells in culture basedupon quantitation of ATP present, which is an indicator of metabolicallyactive cells. For each dose combination used, percent inhibition withrespect to EGF+HRG control is calculated and is given in the upper tableof FIGS. 17A-N. An HSA score is also calculated for each dosecombination, with “True” values (indicating that the activity of thecombination is greater than both of the two therapeutics alone) denotedwith bold font, and “False” values denoted with regular font. For eachdose combination in which both drugs are present, a Bliss independencescore is calculated and is given in the lower table of FIGS. 16A-N.Bliss independence scores less than zero, indicating synergy, aredenoted with bold font, and Bliss independence scores greater than orequal to zero, indicating lack of synergy or additivity, are denotedwith italic and underlined font.

The results shown in FIGS. 16A-N demonstrate that an HSA score of “True”is observed with some combinations of [P1X+P2X+P3X] and MM-121 in allcell lines and is observed with the majority of combinations of[P1X+P2X+P3X] and MM-121 in 12 out of the 14 cell lines assessed. Theresults shown in FIGS. 16A-N also demonstrate that synergy is observedwith some combinations of [P1X+P2X+P3X] and MM-121 in all cell lines andthat synergy is observed with the majority of combinations of[P1X+P2X+P3X] and MM-121 in 12 out of the 14 cell lines assessed.

Example 17 Inhibition of Tumor Cell Proliferation In Vitro byCombinations of [P1X+P2X+P3X] and DTX

Inhibition of tumor cell proliferation in vitro was analyzed in the celllines A549, NCI-H1975, NCI-H226, NCI-H322M, HCC827, HOP-62, SK-MES-1 andSW 900 by the methods described above. Cells were treated with all 80pairwise combinations of [P1X+P2X+P3X] and DTX concentrations listed inTable 17.

TABLE 17 P1X + P2X + P3X Conc, nM DTX Conc, ng/mL 2000 1000 666.7 333.3222.2 111.1 74.1 37.0 24.7 12.3 8.2 4.1 2.7 1.4 0.9 0 0.3 0

It is noted that the concentrations shown in Table 17 are totalconcentrations for P1X+P2X+P3X. The ratio used is 2:2:1, so theindividual concentrations of P1X, P2X and P3X are 40%, 40% and 20% ofthe total concentration, respectively. The ordinarily skilled artisanwill understand that each specific concentration value in Table 17 issubject to some minor experimental variability, so that each specificconcentration given indicates a value of about the indicatedconcentration (e.g., a concentration indicated in a table as 2000 nMrepresents a value of about 2000 nM).

For each cell line, inhibition of cell proliferation was measured asdescribed in Example 16. For each dose combination used, percentinhibition with respect to EGF control is calculated and is given in theupper table of FIGS. 17A-H. An HSA score is also calculated for eachdose combination as described in Example 16, with “True” values denotedwith bold font, and “False” values denoted with regular font. A Blissindependence score is also calculated as described in Example 16 and isshown in the lower table of FIGS. 17A-H. Bliss independence scores lessthan zero, indicating synergy, are denoted with bold font, and Blissindependence scores greater than or equal to zero, indicating lack ofsynergy or additivity, are denoted with italic and underlined font.

The results shown in FIGS. 17A-H demonstrate that an HSA score of “True”is observed with some combinations of [P1X+P2X+P3X] and DTX in all celllines and is observed with the majority of combinations of [P1X+P2X+P3X]and DTX in 7 out of the 8 cell lines assessed. The results shown inFIGS. 17A-H also demonstrate that synergy is observed with somecombinations of [P1X+P2X+P3X] and DTX in all cell lines and that synergyis observed with the majority of combinations of [P1X+P2X+P3X] and DTXin 6 out of the 8 cell lines assessed.

Example 19 Inhibition of Tumor Cell Proliferation In Vitro byCombinations of [P1X+P2X+P3X] and SN-38

Inhibition of tumor cell proliferation in vitro was analyzed in the celllines A549, HCT 116, HT-29 and LoVo by the methods described above. A549and LoVo cells were treated with all 80 pairwise combinations of[P1X+P2X+P3X] and SN-38 concentrations listed in Table 18.

TABLE 18 P1X + P2X + P3X Conc, nM SN-38 Conc, nM 1000 1000 250 250 62.562.5 15.6 15.6 3.9 3.9 0.98 0.98 0.24 0.24 0 00.06 0.02 0

HCT 116 and HT-29 cells were treated with all 48 pairwise combinationsof [P1X+P2X+P3X] and SN-38 concentrations listed in Table 19.

TABLE 19 P1X + P2X + P3X Conc, nM SN-38 Conc, nM 2000 1000 500 250 25062.5 125 15 62.5 3.9 0 1 0.2 0

It is noted that the concentrations shown in Table 18 and 19 are totalconcentrations for P1X+P2X+P3X. The ratio used is 2:2:1, so theindividual concentrations of P1X, P2X and P3X are 40%, 40% and 20% ofthe total concentration, respectively. The ordinarily skilled artisanwill understand that each specific concentration value in Tables 18 and19 is subject to some minor experimental variability, so that eachspecific concentration given indicates a value of about the indicatedconcentration (e.g., a concentration indicated in a table as 2000 nMrepresents a value of about 2000 nM).

For each cell line, inhibition of cell proliferation was measured asdescribed in Example 16. For each dose combination used, percentinhibition with respect to EGF control is calculated and is given in theupper table of FIGS. 18A-D. An HSA score is also calculated for eachdose combination as described in Example 16, with “True” values denotedwith bold font, and “False” values denoted with regular font. A Blissindependence score is also calculated as described in Example 16 and isshown in the lower table of FIGS. 18A-D. Bliss independence scores lessthan zero, indicating synergy, are denoted with bold font, and Blissindependence scores greater than or equal to zero, indicating lack ofsynergy or additivity, are denoted with italic and underlined font.

The results shown in FIGS. 18A-D demonstrate that an HSA score of “True”is observed with the majority of combinations of [P1X+P2X+P3X] and SN-38in all 4 cell lines assessed. The results also demonstrate that synergyis observed with some combinations of [P1X+P2X+P3X] and DTX in all celllines and that synergy is observed with the majority of combinations of[P1X+P2X+P3X] and SN-38 in 2 out of the 4 cell lines assessed.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims. Any combination of the embodimentsdisclosed in the any plurality of the dependent claims is contemplatedto be within the scope of the disclosure.

INCORPORATION BY REFERENCE

All, patents, pending patent applications and patent publicationsreferred to hereinabove are hereby incorporated by reference in theirentireties.

APPENDIX A ANTI-CANCER AGENTS Anti-Cancer Agent Comments ExamplesAntibodies Antibodies which bind A12 (fully humanized mAb) IGF-1R(insulin-like 19D12 (fully humanized mAb) growth factor type 1 CP751-871(fully humanized mAb) receptor), which is H7C10 (humanized mAb)expressed on the cell alphaIR3 (mouse) surface of must human scFV/FC(mouse/human chimera) cancers EM/164 (mouse) AMG 479 (fully humanizedmAb; Amgen) IMCA 12 (fully humanized mAb; Imclone) NSC-742460 (Dyax)MR-0646, F50035 (Pierre Fabre Medicament, Merck) Antibodies which bindmatuzumab (EMD72000) EGFR; Mutations Erbitux ®/cetuximab (Imclone)affecting EGFR Vectibix ®/panitumumab (Amgen) expression or activity canmAb 806 result in cancer nimotuzumab (TheraCIM ®) INCB7839 (Incyte)panitumumab (Vectibix ®; Amgen) Antibodies which bind AV299 (AVEO) cMET(mesenchymal AMG102 (Amgen) epithelial transition 5D5 (OA-5D5)(Genentech) factor); a member of the MET family of receptor tyrosinekinases) Anti-ErbB3 antibodies MM-121 (Merrimack Pharmaceuticals) Ab #14described in WO 2008/100624 1B4C3; 2D1D12 (U3 Pharma AG) U3-1287/AMG888(U3 Pharma/Amgen) Anti-ErbB2 (HER2) Herceptin ® (trastuzumab; antibodiesGenentech/Roche); Omnitarg ® (pertuzumab; 2C4, R1273; Genentech/Roche)Small Molecules IGF-1R (insulin-like NVP-AEW541-A Targeting IGF1R growthfactor type 1 BMS-536,924 (1H-benzoimidazol-2-yl)- receptor), which is1H-pyridin-2-one) expressed on the cell BMS-554,417 surface of musthuman Cycloligan cancers TAE226 PQ401 Small Molecules EGFR; MutationsIressa ®/gefitinib (AstraZeneca) Targeting EGFR affecting EGFR CI-1033(PD 183805) (Pfizer) expression or activity can TYVERB/lapatinib(GlaxoSmithKline) result in cancer Tykerb ®/lapatinib ditosylate(SmithKline Beecham) Tarceva ®/Erlotinib HCL (OSI Pharma) PKI-166(Novartis) PD-158780 EKB-569 Tyrphostin AG 1478(4-(3-Chloroanillino)-6,7-dimethoxyquinazoline) Small Molecules ErbB2, also known as HKI-272(neratinib; Wyeth) Targeting ErbB2 HER2, a member of the KOS-953(tanespimycin; Kosan ErbB family of receptors, Biosciences) which isexpressed on certain cancer cells Small Molecules cMET (MesenchymalPHA665752 Targeting cMET epithelial transition ARQ 197 (ArQule) factor);a member of the ARQ-650RP (ArQule) MET family of receptor tyrosinekinases) Antimetabolites An antimetabolite is a flourouracil (5-FU)chemical with a similar capecitabine/XELODA ® (HLR Roche) structure to asubstance (a 5-trifluoromethyl-2′-deoxyuridine metabolite) required formethotrexate sodium (Trexall) (Barr) normal biochemicalraltitrexed/Tomudex ® (AstraZaneca) reactions, yet differentpemetrexed/Alimta ® (Lilly) enough to interfere with tegafur the normalfunctions of cytosine arabinoside (Cytarabine, Ara-C)/ cells, includingcell tioguanine/Lanvis ® (GlaxoSmithKline) division. 5-azacytidine6-mercaptopurine (Mercaptopurine, 6- MP) azathioprine/Azasan ®(AAIPHARMA LLC) 6-thioguanine (6-TG)/Purinethol ® (TEVA)pentostatin/Nipent ® (Hospira Inc.) fludarabine phosphate/Fludara ®(Bayer Health Care) cladribine/Leustatin ® (2-CdA, 2-chlorodeoxyadenosine) (Ortho Biotech) floxuridine(5-fluoro-2′-deoxyuridine)/ FUDR ® (Hospira, Inc,) Alkylating agents Analkylating Ribonucleotide Reductase Inhibitor antineoplastic agent is an(RNR) alkylating agent that cyclophosphamide/Cytoxan ® (BMS)/ attachesan alkyl group to Neosar ® (TEVA) DNA. Since cancer cellsifosfamide/Mitoxana ® (ASTA Medica) generally proliferate ThioTEPA(Bedford, Abraxis, Teva) unrestrictively more than BCNU→1,3-bis(2-chloroethyl)-1- do healthy cells they are nitosourea moresensitive to DNA CCNU→ 1,-(2-chloroethyl)-3-cyclohexyl- damage, andalkylating 1-nitrosourea (methyl CCNU) agents are used clinicallyhexamethylmelamine (altretamine, HMM)/ to treat a variety of Hexalen ®(MGI Pharma Inc.) tumors. busulfan/Myleran ® (GlaxoSmithKline)procarbazine HCL/Matulane ® (Sigma Tau) Dacarbazine (DTIC ®)chlorambucil/Leukaran ® (SmithKline Beecham) Melphalan/Alkeran ®(GlaxoSmithKline) cisplatin (Cisplatinum, CDDP)/Platinol (Bristol Myers)carboplatin/Paraplatin (BMS) oxaliplatin/Eloxitan ® (Sanofi-Aventis US)Bendamustine carboquone carmustine chloromethine dacarbazine (DTIC)fotemustine lomustine mannosulfan nedaplatin nimustine prednimustineranimustine satraplatin semustine streptozocin temozolomide treosulfantriaziquone triethylene melamine triplatin tetranitrate trofosfamideuramustine Topoisomerase Topoisomerase inhibitors doxorubicinHCL/Doxil ® (Alza) inhibitors are chemotherapy agents daunorubicincitrate/Daunoxome ® designed to interfere with (Gilead) the action ofmitoxantrone HCL/Novantrone (EMD topoisomerase enzymes Serono)(topoisomerase I and II), actinomycin D which are enzymes thatetoposide/Vepesid ® (BMS)/ control the changes in Etopophos ® (Hospira,Bedford, Teva DNA structure by Parenteral, Etc.) catalyzing the breakingtopotecan HCL/Hycamtin ® and rejoining of the (GlaxoSmithKline)phosphodiester backbone teniposide (VM-26)/Vumon ® (BMS) of DNA strandsduring irinotecan HCL(CPT-11)/ the normal cell cycle. camptosar ®(Pharmacia & Upjohn) camptothecin (CPT) belotecan rubitecan MicrotubuleMicrotubules are one of vincristine/Oncovin ® (Lilly) targeting agentsthe components of the vinblastine sulfate/Velban ®(discontinued)cytoskeleton. They have (Lilly) diameter of vinorelbinetartrate/Navelbine ® apporximately 24 nm and (PierreFabre) lengthvarying from vindesine sulphate/Eldisine ® (Lilly) several micrometersto paclitaxel/Taxol ® (BMS) possibly millimeters in docetaxel/Taxotere ®(Sanofi Aventis axons of nerve cells. US) Microtubules serve asNanoparticle paclitaxel (ABI-007)/ structural components Abraxane ®(Abraxis BioScience, Inc.) within cells and are ixabepilone/IXEMPRA ™(BMS) involved in many cellular larotaxel processes including ortataxelmitosis, cytokinesis, and tesetaxel vesicular transport. vinflunineKinase inhibitors Tyrosine kinases are imatinib mesylate/Gleevec(Novartis) enzymes within the cell sunitinib malate/Sutent ® (Pfizer)that function to attach sorafenib tosylate/Nexavar ® (Bayer) phosphategroups to the nilotinib hydrochloride monohydrate/ amino acid tyrosine.By Tasigna ® (Novartis) blocking the ability of AMG 386 (Amgen) proteintyrosine kinases axitinib (AG-013736; Pfizer, Inc.) to function, thesebosutinib (SKI-606; Wyeth) compounds provide a tool brivanib alalinate(BMS-582664; BMS) for controlling cancerous cediranib (AZD2171;Recentin, cell growth. AstraZeneca) dasatinib (BMS-354825: Sprycel ®;BMS) lestaurtinib (CEP-701; Cephalon) motesanib diphosphage (AMG-706;Amgen/Takeda) pazopanib HCL (GW786034; Armala, GSK) semaxanib (SU5416;Pharmacia) vandetanib (AZD647; Zactima; AstraZeneca) vatalanib (PTK-787;Novartis, Bayer Schering Pharma) XL184 (NSC718781; Exelixis, GSK)Protein synthesis Induces cell apoptosis L-asparaginase/Elspar ® (Merck& Co.) inhibitors Immunotherapeutic Induces cancer patients to Alphainterferon agents exhibit immune Angiogenesis Inhibitor/Avastin ®responsiveness (Genentech) IL-2→ Interleukin 2 (Aldesleukin)/Proleukin ® (Chiron) IL-12→ Interleukin 12 Hormonal therapies Hormonaltherapies Ttoremifene citrate/Fareston ® (GTX, associated with Inc.)menopause and aging fulvestrant/Faslodex ® (AstraZeneca) seek toincrease the raloxifene HCL/Evista ® (Lilly) amount of certainanastrazole/Arimidex ® (AstraZeneca) hormones in the bodyletrozole/Femara ® (Novartis) to compensate for age- fadrozole (CGS16949A) or disease-related exemestane/Aromasin ® (Pharmacia & hormonaldeclines. Upjohn) Hormonal therapy as a leuprolide acetate/Eligard ®(QTL USA) cancer treatment Lupron ® (TAP Pharm.) generally eitherreduces goserelin acetate/Zoladex ® the level of one or (AstraZeneca)more specific triptorelin pamoate/Trelstar ® (Watson hormones, blocks aLabs) hormone from buserelin/Suprefact ® (Sanofi Aventis) interactingwith its nafarelin cellular receptor or cetrorelix/Cetrotide ® (EMDSerono) otherwise alters the bicalutamide/Casodex ® (AstraZeneca)cancer's ability to be nilutamide/Nilandron ® (Aventis Pharm.)stimulated by megestrol acetate/Megace ® (BMS) hormones to grow andsomatostatin Analogs (e.g., Octreotide spread. Such hormonalacetate/Sandostatin ® (Novartis)) therapies thus include abarelix(Plenaxis ™; Amgen) hormone antagonists abiraterone acetate (CB7630; BTGplc) and hormone synthesis afimoxifene (TamoGel; Ascend inhibitors. Insome Therapeutics, Inc.) instances hormone aromatase inhibitor(Atamestane plus agonists may also be toremifene; Intarcia Therapeutics,Inc.) used as anticancer arzoxifene (Eli Lilly & Co) hormonal therapies.Asentar ™; DN-101 (Novacea; Oregon Health Sciences U) flutamide(Eulexin ®, Schering; Prostacur, Laboratorios Almirall, S.A) letrozole(CGS20267) (Femara ®, Chugai; Estrochek ®, (Jagsonpal PharmaceuticalsLtd;) Delestrogen ®, estradiol valerate (Jagsonpal) magestrolacetate/Megace ® medroxyprogesteone acetate (Veraplex ®; Combiphar)MT206 (Medisyn Technologies, Inc.) nandrolone decanoate (Zestabolin ®;Mankind Pharma Ltd) tamoxifen (Taxifen ®, Yung Shin Pharmaceutical;Tomifen ®, Alkem Laboratories Ltd.) tamoxifen citrate (Nolvadex,AstraZeneca; soltamox, EUSA Pharma Inc; tamoxifen citrate SOPHARMA,Sopharma JSCo.) Glucocorticoids Anti-inflammatory predinsolone drugsused to reduce dexamethasone/Decadron ® (Wyeth) swelling that causesprednisone (Deltasone, Orasone, Liquid cancer pain. Pred, Sterapred ®)Aromatase inhibitors Includes imidazoles ketoconazole mTOR inhibitorsThe mTOR signaling sirolimus (Rapamycin)/Rapamune ® pathway wasoriginally (Wyeth) discovered during Temsirolimus (CCI-779)/Torisel ®studies of the (Wyeth) immunosuppressive Deforolimus (AP23573) (AriadPharm.) agent rapamycin. This Everolimus (RAD001)/Certican ® highlyconserved (Novartis) pathway regulates cell proliferation and metabolismin response to environmental factors, linking cell growth factorreceptor signaling via phosphoinositide-3- kinase (PI-3K) to cellgrowth, proliferation, and angiogenesis. Chemotherapeutic adriamycin,5-fluorouracil, cytoxin, agents bleomycin, mitomycin C, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins,clofarabine, mercaptopurine, pentostatin, thioguanine, cytarabine,decitabine, floxuridine, gemcitabine (Gemzar), enocitabine, sapacitabineProtein Kinase B AKT Inhibitor Astex ® (Astex (PKB) InhibitorsTherapeutics) AKT Inhibitors NERVIANO (Nerviano Medical Sciences) AKTKinase Inhibitor TELIK (Telik Inc) AKT DECIPHERA (DecipheraPharmaceuticals, LLC) perifosine (KRX0401, D-21266; KeryxBiopharmaceuticals Inc, AEterna Zentaris Inc) perifosine with Docetaxel(Keryx Biopharmaceuticals Inc, AEterna Zentaris Inc) perifosine withGemcitabine (AEterna Zentaris Inc) perifosine with paclitaxel (AEternaZentaris Inc) protein kinase-B inhibitor DEVELOGEN (DeveloGen AG) PX316(Oncothyreon, Inc.) RX0183 (Rexahn Pharmaceuticals Inc) RX0201 (RexahnPharmaceuticals Inc) VQD002 (VioQuest Pharmaceuticals Inc) XL418(Exelixis Inc) ZEN027 (AEterna Zentaris Inc) Phosphatidylinositol BEZ235(Novartis AG) 3-Kinase (PI3K) BGT226 (Novartis AG) Inhibitors CAL101(Calistoga Pharmaceuticals, Inc.) CHR4432 (Chroma Therapeutics Ltd)Erk/PI3K Inhibitors ETERNA (AEterna Zentaris Inc) GDC0941 (GenentechInc/Piramed Limited/Roche Holdings Ltd) enzastaurin HCL (LY317615;Enzastaurin; Eli Lilly) LY294002/Wortmannin PI3K Inhibitors SEMAFORE(Semafore Pharmaceuticals) PX866 (Oncothyreon, Inc.) SF1126 (SemaforePharmaceuticals) VMD-8000 (VM Discovery, Inc.) XL147 (Exelixis Inc)XL147 with XL647 (Exelixis Inc) XL765 (Exelixis Inc) PI-103(Roche/Piramed) Cyclin Dependent CYC200, R-roscovitine (Seliciclib;Kinase Inhibitors Cyclacel Pharma) NSC-649890, L86-8275, HMR-1275(alvocidib; NCI) TLr9, CD289 IMOxine (Merck KGaA) HYB2055 (Idera)IMO-2055 (Isis Pharma) 1018 ISS (Dynavax Technologies/UCSF) PF-3512676(Pfizer) Enzyme Inhibitor lonafarnib(SCH66336; Sarasar; SuperGen, UArizona) Anti-TRAIL AMG-655 (Aeterna Zentaris, Keryx Biopharma)Apo2L/TRAIL, AMG951 (Genentech, Amgen) APOMAB (fully humanized mAb;Genentech) MEK Inhibitors [Mitogen-Activated ARRY162 (Array BioPharmaInc) Protein Kinase Kinase ARRY704 (Array BioPharma Inc) 1 (MAP2K1);Mitogen- ARRY886 (Array BioPharma Inc) Activated Protein AS703026 (MerckSerono S.A) Kinase Kinase 2 AZD6244 (AstraZeneca Plc) (MAP2K2)] AZD8330(AstraZeneca Plc) RDEA119 (Ardea Biosciences, Inc.) RDEA436 (ArdeaBiosciences, Inc.) XL518 (Exelixis Inc; Genentech Inc) MiscellaneousImprime PGG (Biothera) Inhibitors CHR-2797 (AminopeptidaseM1 inhibitor;Chroma Therapeutics) E7820, NSC 719239 (Integrin-alpha2 inhibitor,Eisai) INCB007839 (ADAM 17, TACE Inhibitor; Incyte) CNF2024, BIIB021(Hsp90 Inhibitor; Biogen Idec) MP470, HPK-56 (Kit/Mel/Ret Inhibitor;Schering-Plough) SNDX-275/MS-275 (HDAC Inhibitor; Syndax) Zarnestra ™,Tipifarnib, R115777 (Ras Inhibitor; Janssen Pharma) volociximab; Eos200-4, M200 (alpha581 integrin inhibitor; Biogen Idec; EliLilly/UCSF/PDL BioPharma) apricoxib (TP2001; COX-2 Inhibitor, DaiichiSankyo; Tragara Pharma)

SEQUENCE LISTING SUMMARY P1X V_(H) CDR1 SYAIS SEQ ID NO: 1P1X V_(H) CDR2 IIPIFGTVNY SEQ ID NO: 2 P1X V_(H) CDR3 DPSVNLSEQ ID NO: 3 P1X V_(L) CDR1 QSISSWWA SEQ ID NO: 4 P1X V_(L) CDR2 DASSLSEQ ID NO: 5 P1X V_(L) CDR3 QQYHAHP SEQ ID NO: 6 P2X V_(H) CDR1 SYAISSEQ ID NO: 7 P2X V_(H) CDR2 IIPIFGAANP SEQ ID NO: 8 P2X V_(H) CDR3MGRGKV SEQ ID NO: 9 P2X V_(L) CDR1 QSVLYSPNNKNYLA SEQ ID NO: 10P2X V_(L) CDR2 WASTR SEQ ID NO: 11 P2X V_(L) CDR3 QQYYGSP SEQ ID NO: 12P3X V_(H) CDR1 SYGIN SEQ ID NO: 13 P3X V_(H) CDR2 ISAYNGNTYYSEQ ID NO: 14 P3X V_(H) CDR3 DLGGYGSGS SEQ ID NO: 15 P3X V_(L) CDR1QSVSSNLA SEQ ID NO: 16 P3X V_(L) CDR2 GASTR SEQ ID NO: 17 P3X V_(L) CDR3QDYRTWPR SEQ ID NO: 18 P1X V_(H) MGFGLSWLFLVAILKGVQC SEQ ID NO: 19QVQLVQSGAEVKKPGSSVKV SCKASGGTFSSYAISWVRQA PGQGLEWMGSIIPIFGTVNYAQKFQGRVTITADESTSTAY MELSSLRSEDTAVYYCARDP SVNLYWYFDLWGRGTLVTVSSP1X V_(L) MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 20 DIQMTQSPST LSASVGDRVTITCRASQSISSWWAWYQQKP GKAPKLLIYDASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQYHAHPTTFGG GTKVEIK P2X V_(H) MGFGLSWLFLVAILKGVQC SEQ ID NO: 21QVQLVQSGAEVKKPGSSVKV SCKASGGTFGSYAISWVRQA PGQGLEWMGSIIPIFGAANPAQKSQGRVTITADESTSTAY MELSSLRSEDTAVYYCAKMG RGKVAFDIWGQGTMVTVSS P2X V_(L)MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 22 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSPNNKNYLA WYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGS PITFGGGTKVEIK P3X V_(H) MGFGLSWLFLVAILKGVQCSEQ ID NO: 23 QVQLVQSGAEVKKPGASVKV SCKASGYAFTSYGINWVRQAPGQGLEWMGWISAYNGNTYY AQKLRGRVTMTTDTSTSTAY MELRSLRSDDTAVYYCARDLGGYGSGSVPFDPWGQGTLVTVSS P3X V_(L) MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 24EIVMTQSPATLSVSPGERAT LSCRASQSVSSNLAWYQQKP GQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQDYRTWPRRVFG GGTKVEIK pMP10K_IgG1RTVAAPSVFIFPPSDEQLKS SEQ ID NO: 25 Light Chain GTASVVCLLNNFYPREAKVQKappa- WKVDNALQSGNSQESVTEQD Constant SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC pMP10K_IgG1 ASTKGPSVFPLAPSSKSTSGSEQ ID NO: 26 Heavy Chain GTAALGCLVKDYFPEPVTVS (EEM)_WNSGALTSGVHTFPAVLQSS Constant GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK ca/cd V_(H) CDR2IIPIFGTANY SEQ ID NO: 27 ca V_(H) CDR3 DPSVDL SEQ ID NO: 28ca V_(L) CDR1 QSISSWLA SEQ ID NO: 29 ca V_(L) CDR3 QQFAAHA SEQ ID NO: 30cd V_(L) CDR1 QSVLYSSNNKNYLA SEQ ID NO: 31 ch V_(H) CDR2 ISAYNGNTNYSEQ ID NO: 32

EGFR ECD (SEQ ID NO: 33) 1MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV 61VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA 121VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF 181QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC 241TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV 301VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK 361NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF 421ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL 481FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCN 541LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM 601GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSHHHHH H

What is claimed is:
 1. A monoclonal antibody which binds EGFRextracellular domain and comprises heavy and light chain CDR1, CDR2, andCDR3 sequences, wherein the heavy and light chain CDR1, CDR2, and CDR3sequences are selected from the group consisting of: (a) heavy chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3 respectively,and light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and6, respectively; (b) heavy chain CDR1, CDR2, and CDR3 sequences of SEQID NOs: 7, 8, and 9, respectively, and light chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 10, 11 and 12, respectively; and (c) heavychain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 13, 14, and 15,respectively, and light chain CDR1, CDR2, and CDR3 sequences of SEQ IDNOs: 16, 17, and 18, respectively.
 2. A monoclonal antibody that bindsto EGFR extracellular domain and comprises a heavy chain variable regionand a light chain variable region, wherein the heavy and light chainvariable region sequences are selected from the group consisting of: (a)a heavy chain variable region comprising SEQ ID NO: 19 and a light chainvariable region comprising SEQ ID NO: 20; (b) a heavy chain variableregion comprising SEQ ID NO: 21 and a light chain variable regioncomprising SEQ ID NO: 22; and (c) a heavy chain variable regioncomprising SEQ ID NO: 23 and a light chain variable region comprisingSEQ ID NO:
 24. 3. The monoclonal antibody of claim 1, which binds toEGFR with a K_(D) of better than 100 nM.
 4. The monoclonal antibody ofclaim 1, which is a human antibody.
 5. The monoclonal antibody of claim1, wherein the antibody is selected from the group consisting of abispecific antibody, immunoconjugate, Fab, Fab′2, ScFv, avimer, nanobodyand a domain antibody.
 6. The monoclonal antibody of claim 1, whereinthe monoclonal antibody is selected from the group consisting of IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD and IgE isotypeantibodies.
 7. A pharmaceutical composition comprising the monoclonalantibody of claim 1 and a pharmaceutically acceptable carrier.
 8. A kitcomprising the pharmaceutical composition of claim 7 in a container. 9.The monoclonal antibody of claim 1 for the treatment of a cancer.
 10. Acomposition comprising two or three monoclonal antibodies which bind toEGFR extracellular domain, wherein the two or three monoclonalantibodies are selected from the group consisting of: (a) a monoclonalantibody comprising heavy chain CDR1, CDR2, and CDR3 sequences of SEQ IDNOs: 1, 2, and 3 respectively, and light chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 4, 5, and 6, respectively; (b) a monoclonalantibody comprising heavy chain CDR1, CDR2, and CDR3 sequences of SEQ IDNOs: 7, 8, and 9, respectively, and light chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 10, 11 and 12, respectively and (c) amonoclonal antibody comprising heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 13, 14, and 15, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17 and 18,respectively; and wherein the composition comprises (a) and (b), (a) and(c), (b) and (c) or (a), (b) and (c).
 11. A composition comprising twoor three monoclonal antibodies which bind to EGFR extracellular domain,wherein the two or three monoclonal antibodies are selected from thegroup consisting of: (a) a monoclonal antibody comprising a heavy chainvariable region comprising SEQ ID NO: 19 and a light chain variableregion comprising SEQ ID NO: 20; (b) a monoclonal antibody comprising aheavy chain variable region comprising SEQ ID NO: 21 and a light chainvariable region comprising SEQ ID NO: 22; and (c) a monoclonal antibodycomprising a heavy chain variable region comprising SEQ ID NO: 23 and alight chain variable region comprising SEQ ID NO: 24; and wherein thecomposition comprises (a) and (b), (a) and (c), (b) and (c) or (a) (b)and (c).
 12. The composition of claim 10, wherein each of monoclonalantibodies (a), (b) and (c) binds to EGFR with a K_(D) of better than100 nM.
 13. The composition of claim 12, wherein each of monoclonalantibodies (a), (b) and (c) binds to EGFR with a K_(D) of better than 10nM.
 14. The composition of claim 10, wherein each of monoclonalantibodies (a), (b) and (c) is a human antibody.
 15. The composition ofclaim 10, wherein one or more of monoclonal antibodies (a), (b), and (c)is independently selected from the group consisting of a bispecificantibody, immunoconjugate, Fab, Fab′2, ScFv, avimer, nanobody and adomain antibody.
 16. The composition of claim 10, wherein each ofmonoclonal antibodies (a), (b), and (c) is independently selected fromthe group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec,IgD and IgE isotype antibodies.
 17. The composition of claim 10, whichfurther comprises a pharmaceutically acceptable carrier.
 18. A kitcomprising the composition of claim 17 in a container.
 19. Thecomposition of claim 10 for the treatment of a cancer.
 20. A compositioncomprising three monoclonal anti-EGFR antibodies, said compositioncomprising a first antibody, a second antibody and a third antibody,wherein (i) the first antibody comprises heavy chain CDR1, CDR2, andCDR3 sequences of SEQ ID NOs: 1, 2, and 3 respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively;(ii) the second antibody comprises heavy chain CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11 and 12,respectively; and (iii) the third antibody comprises heavy chain CDR1,CDR2, and CDR3 sequences of SEQ ID NOs: 13, 14, and 15 respectively, andlight chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 16, 17, and18, respectively, and wherein the first second and third antibodies arepresent at a molar ratio of 2:2:1 to each other.
 21. A compositioncomprising three monoclonal anti-EGFR antibodies, said compositioncomprising a first antibody, a second antibody and a third antibody,wherein (i) the first antibody comprises a heavy chain variable regioncomprising SEQ ID NO: 19 and a light chain variable region comprisingSEQ ID NO: 20; (ii) the second antibody comprises a heavy chain variableregion comprising SEQ ID NO: 21 and a light chain variable regioncomprising SEQ ID NO: 22; and (iii) the third antibody comprises a heavychain variable region comprising SEQ ID NO: 23 and a light chainvariable region comprising SEQ ID NO: 24, and wherein the first secondand third antibodies are present at a molar ratio of 2:2:1 to eachother.
 22. The composition of claim 20, wherein each of the firstantibody, the second antibody and the third antibody binds to EGFR witha K_(D) of better than 100 nM.
 23. The composition of claim 20, whereinthe first antibody binds to EGFR with a K_(D) in a range of 1×10⁻⁹ M to1.1×10⁻¹¹ M, the second antibody binds to EGFR with a K_(D) in a rangeof 1×10⁻⁹ M to 7.0×10⁻¹¹ M and the third antibody binds to EGFR with aK_(D) in a range of 1×10⁻⁹ M to 3.6×10⁻¹⁰ M.
 24. The composition ofclaim 20, wherein each of the first antibody, the second antibody andthe third antibody are human antibodies.
 25. The composition of claim20, wherein one or more of the first antibody, the second antibody andthe third antibody are independently selected from the group consistingof a bispecific antibody, immunoconjugate, Fab, Fab′2, ScFv, avimer,nanobody and a domain antibody.
 26. The composition of claim 20, whereineach of the first antibody, the second antibody and the third antibodyis independently selected from the group consisting of IgG1, IgG2, IgG3,IgG4, IgM, IgA1, IgA2, IgAsec, IgD and IgE isotype antibodies.
 27. Thecomposition of claim 20, which further comprises a pharmaceuticallyacceptable carrier.
 28. The composition of claim 27, which is a sterilecomposition and/or suitable for injection.
 29. The composition of claim27, which is a sterile composition suitable for intravenous injection.30. A kit comprising the composition of claim 27 in a container.
 31. Thecomposition of claim 20 for the treatment of a cancer.
 32. Themonoclonal antibody of claim 1, wherein the heavy chain CDR1, CDR2, andCDR3 sequences comprise SEQ ID NOs: 1, 2, and 3 respectively, and thelight chain CDR1, CDR2 and CDR3 sequences comprise SEQ ID NOs: 4, 5, and6, respectively.
 33. The monoclonal antibody of claim 1, wherein theheavy chain CDR1, CDR2, and CDR3 sequences comprise SEQ ID NOs: 7, 8,and 9, respectively, and the light chain CDR1, CDR2, and CDR3 sequencescomprise SEQ ID NOs: 10, 11 and 12, respectively.
 34. The monoclonalantibody of claim 1, wherein the heavy chain CDR1, CDR2, and CDR3sequences comprise SEQ ID NOs: 13, 14, and 15, respectively, and thelight chain CDR1, CDR2, and CDR3 sequences comprise SEQ ID NOs: 16, 17,and 18, respectively.
 35. The monoclonal antibody of claim 1, whereinthe heavy chain variable region comprises SEQ ID NO: 19 and the lightchain variable region comprises SEQ ID NO:
 20. 36. The monoclonalantibody of claim 1, wherein the heavy chain variable region comprisesSEQ ID NO: 21 and the light chain variable region comprises SEQ ID NO:22.
 37. The monoclonal antibody of claim 1, wherein the heavy chainvariable region comprises SEQ ID NO: 23 and the light chain variableregion comprises SEQ ID NO: 24.